THE REGULATION OF SECRETORY CLUSTERIN EXPRESSION
AFTER IONIZING RADIATION EXPOSURE
By
TRACY CRISWELL
Submitted in partial fulfillment of the requirements
For the degree of Doctor of Philosophy
Thesis Advisor: Dr. David A. Boothman
Program in Molecular and Cellular Basis of Disease
Department of Pathology
CASE WESTERN RESERVE UNIVERSITY
May 2004 CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the dissertation of
______
candidate for the Ph.D. degree *.
(signed)______(chair of the committee)
______
______
______
______
______
(date) ______
*We also certify that written approval has been obtained for any proprietary material contained therein. Dedication
This thesis is dedicated to my family, especially my husband, Jamie, my children,
McKenzie and Gaelen and my parents, Linda and Thomas Flamm. I would not be the
person I am today without their perpetual love and support. 2 Table of Contents
Title Page 1
Signature Sheet 2
Dedication 3
Table of Contents 4
List of Tables 5
List of Figures 6-7
Acknowledgements 8
List of Abbreviations 9-10
Abstract 11-12
CHAPTER 1: Introduction
1.1: Clusterin 13-21
1.2: Transcription Factors Activated in Mammalian Cells
After Clinically Relevant Doses of Ionizing Radiation 22-56
CHAPTER 2: Repression of IR-Inducible Clusterin
Expression by the p53 Tumor Suppressor Protein 57-88
CHAPTER 3: Induction of Clusterin, a Pro-Survival Factor,
Requires a Delayed Activation of the IGF-1R/MAPK
Signal Cascade After IR 89-124
CHAPTER 4: Conclusions and Future Directions 125-134
BIBLIOGRAPHY 135-186
3 List of Tables
Table 1.1. Various names assigned to clusterin
Table 1.2. IR-inducible transcription factors
Table 2.1. sCLU is a stress protein induced by a variety of cytotoxic agents
Table 2.2. Effect of p53 status on sCLU basal and IR-inducible expression
4 List of Figures
Figure 1.1. Schematic of clusterin gene structure
Figure 1.2. A schematic diagramming the processing of the CLU message into the
various CLU protein forms
Figure 1.3. DNA damage resulting from ionizing radiation exposure results in activation
of signaling cascades that lead to either cell survival or apoptosis
Figure 1.4. IR stimulates the phosphorylation of IKK, which results in the degradation of
IκB allowing for NF-κB to move into the nucleus and activate downstream
Figure 2.1. sCLU is transcriptionally upregulated after IR exposure in MCF-7 human
breast cancer cells
Figure 2.2. sCLU basal levels are elevated in MCF-7 and RKO cells that over-express the
HPV E6 protein
Figure 2.3. sCLU is induced in HCT116:p53-/- cells, but not in p53-/- HCT116 parental
cells
Figure 2.4. sCLU is not induced in HCT116:p21-/- cells
Figure 2.5. sCLU is not cell cycle regulated
Figure 3.1. siRNA to sCLU in MCF-7 cells results in radio-sensitization
Figure 3.2. Activation of MAPK by IR
Figure 3.3. EGFR is not an upstream activator of sCLU
5 Figure 3.4. Inhibition of Insulin-like Growth Factor-1 Receptor (IGF-1R) abrogates
sCLU induction after IR
Figure 3.5. c-Src is an upstream activator of sCLU induction after IR
Figure 3.6. Activation of the MAPK cascade is required for sCLU induction after IR
Figure 3.7. The Egr-1 transcription factor can bind to the CLU promoter and is required
for promoter activation after IR
Figure 4.1. Model indicating hypothetical cross-talk between the signals for sCLU
induction after IR and p53 repression.
6 Acknowledgements
I would like to acknowledge everyone who has helped me through the past several years during my graduate career.
First I would like to thank my mentor, David A. Boothman, Ph.D who has guided me when I needed it and left me to find my own path when I needed to do that as well.
You encouraged me from the start and introduced me to a scientific community that I would not have found on my own. I would also like to thank Dr. Lindsey Mayo who has guided me through the clusterin signaling maze over the past year.
I would also like to thank my committee members, Dr. Alan Tartakoff, Dr. Sanjay
Pimplikar, Dr. Susann Brady-Kalnay, Dr. Lindsey Mayo and Dr. Clive Hamlin for helping me to “stay on the straight and narrow” path. Their guidance helped to keep me focused and their support helped me to become the scientist that I am today.
Thank you to everyone in the Boothman lab, both past and present. You have been my company and my friends for the past several years, and the relationships I have made have helped me through the rough times.
Lastly, I want to thank my husband, Jamie, and my children, McKenzie and
Gaelen. Thank you for your never ending support and love. Thank you for putting up with my work on the weekends and evenings. Thank you for lifting me up when I was low and letting me rest when I was tired, and thank you for being there every step of the way.
7 List of Abbreviations
ATM ataxia telengiectasia mutated
ATR ataxia telengiectasia related gene
BASC BRCA-containing assembly complex bp base pair
CDK cyclin-dependent kinase
CLU clusterin
CMV cytomegalovirus dn dominant negative
DSB DNA double-strand break
EGF epidermal growth factor
EGFR epidermal growth factor receptor
EMSA electrophoretic mobility shift assay
ER endoplasmic reticulum
FBS fetal bovine serum
HUVEC human umbilical vein endothelial cells
IEGs immediate early genes
IGF-1 insulin-like growth factor-1
IGF-1R insulin like growth factor-1 receptor
IGFBPs insulin-like growth factor binding proteins
IR ionizing radiation
MAPK mitogen activated protein kinase
8 NAC n-acetylcysteine nCLU nuclear clusterin
NLS nuclear localization sequence
PDT photodynamic therapy
PI propidium iodide psCLU pre-secretory clusterin
RCEs retinoblastoma control elements
RCPs retinoblastoma control proteins
ROS reactive oxygen species
SAPK stress activated protein kinase sCLU secretory clusterin
SERCA sarco-endoplasmic reticulum calcium ATPase
Src CA constitutively active Src
Src KD kinase dead Src
SREs serum response elements
SSBs DNA single-strand breaks
TG thapsigargin
TPA tetradecanoylphorbol acetate
UV ultraviolet radiation
VWF von Willebrand factor wt wild-type xip x-ray induced protein
9 The Regulation of Secretory Clusterin Expression after Ionizing
Radiation Exposure
Abstract
By
TRACY CRISWELL
Radiation therapy is a common treatment for many types of tumors. Therefore, it is vital to understand the cellular responses to radiotherapy in malignant cells, as well as the surrounding normal tissues for optimizing the efficacy of this therapy. Our laboratory identified clusterin (CLU) as a protein/transcript that could be induced by doses of ionizing radiation (IR) as low as 0.02 Gy, suggesting a role for CLU in the cellular response to IR. CLU is a secreted glycoprotein that has been implicated in many normal biological processes as well as many pathological processes (Chapter 1). While the exact function of CLU still remains unknown, it has been suggested that secretory CLU
(sCLU), the fully processed and glycosylated form of CLU, plays a role in cytoprotection after cellular stress.
This thesis characterizes the regulation of sCLU after IR exposure. We observed that sCLU protein levels were low and only marginally induced after IR in cell lines containing functional wild-type p53. In contrast, cell lines expressing mutant p53 or null p53 had high basal levels of sCLU. This led us to investigate the role of p53 as a transcriptional repressor of sCLU (Chapter 2). Using genetically matched cell lines, we demonstrated that sCLU is transcriptionally repressed by p53.
10 Further work was done to characterize the signaling pathways required for sCLU induction after IR. We demonstrated that sCLU is involved in cell survival after IR using sCLU specific siRNA (Chapter 3). Furthermore, we demonstrated that the Src-Mek-Erk signaling cascade is reactivated 24-72 h after IR, which corresponds to the late induction of sCLU, and that the signal for sCLU induction after IR is generated by activation of the insulin-like growth factor-1 receptor (IGF-1R). Finally, we demonstrate that this signaling cascade culminates in the activation of Egr-1, which is required for the induction of sCLU after IR.
11 Chapter 1: Introduction
1.1 Clusterin
Clusterin (CLU) is a secreted glycoprotein originally identified in 1983 as a protein involved in cellular aggregation in ram rete testis fluid (1, 2). In 1987, Leger et al. demonstrated that CLU was induced in the ventral rat prostate after castration and named it testosterone-repressed prostate message 2 (TRPM-2) (3). Since then, many groups working in diverse fields have identified/cloned CLU, resulting in diverse nomenclature for this protein (Table 1.1) (1, 3-11). CLU has been implicated in many physiological processes such as lipid metabolism (12, 13), complement regulation (10,
14, 15), cell differentiation (16, 17), reproduction (1, 18, 19), tissue remodeling/regeneration (20-25), cell adhesion (26-28) and cell death (29-35). CLU has also been implicated in many diverse disease processes including atherosclerosis (36-38),
Alzheimer’s disease (39-41), glomerulonephritis (24, 42, 43), preeclampsia (44), lupus erythematosus (45), retinitis pigmentosa (46, 47) and scrapie (29).
Elevated levels of secretory CLU (sCLU) protein and mRNA have been observed in several different types of human neoplasias and malignancies including prostate cancer
(48-50), pancreatic cancer (51), hemangiomas (52), malignant lymphomas (53-55), ovarian cancer (56, 57), breast cancer (58), colorectal carcinoma (59) and renal clear cell carcinomas (60, 61). Additionally, it has been shown that forced over-expression of sCLU in transformed cell lines resulted in an increased resistance to doxorubicin, cisplatin and taxol (48, 62, 63) and abrogation of CLU mRNA expression following
12 Table 1.1
Various names assigned to clusterin and the species from which it was isolated.
Name Species Sample Type Association Year Ref Clusterin ram rete testes fluid reproduction 1983 (1, 2) Glycoprotein III (GP-III) bovine adrenal medulla chromaffin granules 1984 (6) Sulfated glycoprotein-2 (SGP-2) rat sertoli cells reproduction 1984 (64) Testosterone-repressed prostate message 2 (TRPM2) rat prostate apoptosis 1987 (3) gp80 canine renal cells vectorial secretion 1987 (7) SP-40,40 human serum complement regulation 1988 (18) pTB16 human brain gliomas and epileptic foci 1988 (65) T64 quail neuroretinal cells cell transformation 1989 (66) complement lysis inhibitor (CLI) human serum complement regulation 1989 (9) SGP-2 hamster brain scrapie 1989 (20) pADHC-9 human brain Alzheimer’s disease 1989 (67) X-ray induced protein-8 (XIP-8) human melanoma cells IR induction 1989 (11) Apolipoprotein J (Apo-J) human blood lipid transport 1990 (68) Clusterin rat spermatozoa reproduction 1991 (8, 18) NA1/NA2 human blood lipid transport 1991 (8) K611 human retina Retinitis pigmentosa 1992 (69) pc38k porcine vascular smooth muscle nodule formation 1992 (70) Ku70 binding protein 1 (KUB 1) human breast epithelial cells Ku70 binding protein 1999 (71)
13 antisense expression lead to modest chemo- and IR-sensitization in various cell lines (62,
72-74). These data support a cytoprotective role for sCLU and suggest that over- expression of endogenous CLU provides a survival advantage for the tumors in which it is expressed.
The function of CLU still remains to be elucidated. The defining property of
CLU seems to be that it is induced under conditions that result in cellular stress. This fits with its proposed role as a molecular chaperone involved in the clearance of cell debris after cellular stress (75-78). Additionally, Humphreys et al., demonstrated that CLU has properties similar to small heat shock proteins (75), and that sCLU can protect the proteins S-transferase and catalase from heat-induced precipitation, due to improper protein folding, through the formation of high molecular weight complexes (75).
Furthermore, Poon et al. demonstrated that the ability of CLU to protect cells from precipitation was maximal at a slightly acidic pH (79). This suggests a role for sCLU at sites of injury and inflammation, where the pH is slightly acidic to prevent against infection.
The development of a CLU knock-out mouse has allowed further insight into the function of this protein (80). The CLU knock-out mice develop a more severe myocarditis, compared to wild-type mice, with substantial scarring after challenge with murine myosin (80), and Han et al., demonstrated that CLU deficient mice had 50% less cell death after hypoxic/ischemic insult (81). Additionally, Rosenberg et al., showed that
CLU deficient mice had increased immune complex deposition in the kidneys, indicative of progressive glomerulopathy (82). These data suggest a protective role for CLU during tissue injury and inflammatory responses. Additionally, CLU (also termed
14 apolipoprotein J) and apolipoprotein E (ApoE) double knock-out mice showed a decrease in deposition of fibrillar β-amyloid in the brain (83), supporting a role for CLU in
Alzheimer’s disease and diseases of aging.
The signaling pathways that result in CLU induction after stress have not been elucidated. In 1992, Herrault et al., found that v-Src could induce transcription of the avian CLU gene (69). The involvement of Src in human CLU induction has not been investigated. TGF-β also appears to play a role in CLU gene and protein expression (84,
85), potentially through c-Fos binding to an Ap-1 site located in the CLU promoter (86).
Additionally, extracellular CLU can bind to the TGF-β receptor II, suggesting a possible feedback mechanism where CLU affects the downstream TGF-β signaling pathways.
The role of CLU in TGF-β mediated signaling pathways has not been fully elucidated.
B-Myb has also been shown to bind and activate the CLU promoter (87), but the physiologic relevance of this has yet to be determined.
NF-κB has also been shown to regulate sCLU expression. Saura et al., demonstrate that NF-κB is required for LPS stimulation of sCLU expression in glial cells
(88) and Li et al., using microarray technology, demonstrate that TNF-stimulated CLU expression is dependent on the NF-κB/IKK complex (89). Of interest, Santilli et al., demonstrate that sCLU can disrupt NF-κB signaling by stabilizing the inhibitors of NF-
κB (IκBs) (90). This suggests a possible negative feedback loop, where NF-κB stimulates the expression of sCLU after LPS or TNF-α exposure that then acts to stabilize IκBs and silence NF-κB signaling. Signaling after IR has not been elucidated.
Our laboratory isolated CLU as an X-ray-inducible protein/transcript (xip8), in which northern blot and nuclear run-on studies demonstrated enhanced transcript
15 synthesis and steady state mRNA accumulation within IR-exposed human malignant melanoma cells (11, 91, 92). We further identified CLU as a Ku70 binding partner (KUB
1) through a yeast two hybrid screen using Ku70 as bait. Since Ku70 is predominantly localized to the nucleus, we proposed a nuclear form of the CLU protein (nCLU). We found that a 49 kDa nCLU protein resided in the cytoplasm of unirradiated MCF-7 cells and that after IR, this form translocated to the nucleus as a 55 kDa protein (35). The post-translational modifications that result in this change in molecular weight are currently being investigated.
The human CLU mRNA contains two AUG start sites separated by 32 amino acids – therefore one message appears to encode two separate proteins, whose processing is dictated by the presence (sCLU) or absence (nCLU) of this leader peptide sequence
(19, 93) (Figure 1.1). When the CLU mRNA is read at its first AUG sequence, a leader peptide targets the protein to the endoplasmic reticulum (ER). This 60 kDa unmodified peptide (pre-sCLU) is cleaved at an α/β cleavage site to yield two 40 kDa subunits that heterodimerize through the formation of five disulfide bonds to form the mature 80 kDa secretory form of the protein that is further processed and glycosylated in the golgi apparatus (Figure 1.2) (5, 19). sCLU separates as an 80 kDa protein or ~40 kDa proteins under SDS-PAGE non-reducing or reducing conditions, respectively (35). Treatment of log-phase MCF-7 cells with ≥ 2 cGy (2 rads) of ionizing radiation (IR) results in dramatic increases in production of sCLU message and protein, as well as a massive accumulation of the 60 kDa peptide that is presumably present in the ER and golgi of IR-exposed cells within 24-72 h post-IR exposure (35, 94).
16 ATG ATG
NLS N C Coiled-coil Coiled-coil domain domain aa 42-98 aa 319-349 α/β cleavage site
Figure 1.1. Schematic of CLU gene structure. Red hashed boxes correspond to the two coiled –coil domains at amino acids 42-98 and 319-349 respectively (95). Black boxes correspond to two potential nuclear localization sequences.
17 We recently noted that, due to alternative splicing of the CLU mRNA, CLU can be translated from the second AUG start site, resulting in the formation of a 49 kDa pre- nuclear (pre-nCLU) protein observed in the cytoplasm of control non-irradiated MCF-7 human breast cancer cells (95). The nCLU protein contains two putative nuclear localization signals (NLS); the first one located after the second AUG start site and the second located at the C-terminus (Figure 1.1). We propose that these NLSs are kept concealed either through homodimerization or protein folding, through the two coiled- coil domains, until after cellular stress (95). After treatment of log-phase MCF-7 cells with as little as 1.0 Gy of IR, the levels of pre-nCLU protein, as well as a ~55 kDa nuclear form of the protein (nCLU), dramatically increased 48 to 72 h (35, 71). Analyses of nCLU (isolated from the nuclei of IR-exposed MCF-7 cells) revealed that the protein was not cleaved at its α/ß site, since its migration was not altered under reducing or non- reducing SDS-PAGE conditions. Furthermore, accumulation of nCLU protein appears to be sufficient for signaling cell death, even in the absence of IR exposure, and the protein appears to associate with the Ku70/Ku80 DNA double strand break repair machinery
(35).
18 NLS
Clusterin AUG AUG mRNA
Leader Peptide NLS 60 kDa Unmodified α β Precursor α/β cleavage Form (psCLU) Glycosylation
80 kDa
α S S S S S Modified S S S S S Secretory β Form (sCLU)
Figure 1.2. The human CLU mRNA contains two AUG start sites separated by 32 amino acids. When the CLU mRNA is translated from its first AUG sequence, a leader peptide targets the protein to the endoplasmic reticulum (ER) as it is being translated. This 60 kDa unmodified peptide (psCLU) is cleaved at an α/β cleavage site to yield two 40 kDa subunits that heterodimerize through the formation of 5 disulfide bonds to form the 80 kDa secretory form of the protein that is highly glycosylated in the golgi apparatus. Translation from the second AUG start site results in the production of the nuclear form of CLU (nCLU).
19 In summary, CLU is a protein implicated in many normal biological processes and many pathological disease processes. The function of sCLU still remains to be elucidated, but it seems to play a role in cellular stress responses. sCLU appears to provide cytoprotection against cellular injury and inflammatory responses while the nuclear form of the protein appears to be cytotoxic. The regulation of this protein after stress is not understood. A better understanding of this protein and its various roles in cellular responses to stress will allow us to generate better treatments and therapies for many of these different pathological processes.
20 1.2: Transcription factors activated in mammalian cells after clinically relevant
doses of ionizing radiation
This work was published in Oncogene
Oncogene 2003 Sept 1; 22; 5813-5827
Abstract
Over the past fifteen years, a wealth of information has been published on transcripts and proteins ‘induced’ (requiring new synthesis) in mammalian cells after ionizing radiation
(IR) exposure. Many of these studies have also attempted to elucidate the transcription factors that are ‘activated’ (i.e., not requiring de novo synthesis) in specific cells by IR.
Unfortunately, all too often this information has been obtained using supra-lethal doses of
IR, with investigators assuming that induction of these proteins, or activation of corresponding transcription factors, can be “extrapolated” to low-dose IR exposures.
This chapter focuses on what is known at the molecular level about transcription factors induced at clinically relevant (<2 Gy) doses of IR. A review of the literature demonstrates that extrapolation from high to low doses of IR is inaccurate for most transcription factors and most IR-inducible transcripts/proteins, and that induction of transactivating proteins at low doses must be empirically derived. The signal transduction pathways stimulated after high versus low doses of IR, which act to transactivate certain transcription factors in the cell, will be discussed. To date, only three transcription factors appear to be responsive (i.e., activated) after physiological doses (doses wherein cells survive or recover) of IR. These are p53, NF-κB and the Sp1- related retinoblastoma control proteins (RCPs) and related Egr-1 transcription factors.
21 Clearly, more information on transcription factors and proteins induced in mammalian cells at clinically or environmentally relevant doses of IR is needed to understand the role of these stress responses in cancer susceptibility/resistance and radio- sensitivity/resistance mechanisms.
IR-inducible responses in mammalian cells
Ionizing radiation exposure of mammalian cells causes a spectrum of lesions within the cell. At the DNA level, these lesions include DNA single- and double-strand breaks (SSBs, DSBs, respectively), DNA base damage and apyrimidinic/apurinic sites and DNA-protein cross-links. Although the most important lesion for triggering cell death is the formation of DSBs, other DNA lesions (e.g., formation of 8-hydroxyguanine) may have dramatic mutagenic and carcinogenic, consequences (96). Aside from DNA damage, IR also causes a spectrum of other lesions in cellular macromolecules (e.g., lipid peroxidation) due to the generation of reactive oxygen species (ROSs) created by the ionization of water and iron-related Fenton reactions within the cell. Although these non-
DNA lesions are probably not lethal to the cell, they can stimulate various signal transductions pathways (such as protein kinase C (PKC), JNK, ceramide, and MAPK activation) after certain doses of IR. Thus, IR causes a spectrum of DNA and non-DNA lesions that represent potential signals that activate sensory proteins (97). These sensory proteins act to signal that damage has occurred in the cell, as well as regulate processes that both halt cell cycle progression and stimulate repair of DNA lesions.
22 Signals from DNA
At the DNA level, damage sensors stimulated by IR include: (a) the DNA- dependent protein kinase repair complex (DNA-PK, composed of Ku70, Ku80 and DNA-
PK catalytic subunit [DNA-PKcs]) (98, 99); (b) the ataxia telengiectasia (AT) mutated gene (ATM)/c-ABL complex (100, 101); (c) the ataxia telangiectasia-related gene (ATR)
(102, 103); (d) the NBS1-MRE-11-Rad50 complex (104); (e) the DNA mismatch repair complex (97, 105-107); and (f) the Rad9-Hus1-Rad-1 (9:1:1) complex (108-110), among others as yet to be defined processes within mammalian cells. It may be possible that many of these damage sensors exist in a huge complex, referred to as the BRCA- containing assembly complex (BASC complex), and are activated based on the type and/or extent of DNA damage (111) (Figure 1.3).
These damage sensors not only play a role in DNA repair, but also simultaneously regulate cell cycle checkpoint responses that control life and death decisions. Eventually, the damage sensors stimulate specific signal transduction processes that activate specific transcription factors, such as Sp-1, p53, and NF-κB. These activated transcription factors, in turn, control gene expression, cell cycle checkpoint regulation, and under severe conditions may stimulate apoptosis. In recent years, our knowledge of the circuitry of these pathways has increased dramatically, mostly from the development of sound genetic models. We are only now beginning to understand the significance of these pathways in cell cycle progression, apoptosis, survival, mutagenesis and carcinogenesis (particularly when these pathways are altered due to accumulated or inherited mutations). However, the promise of completely understanding the retrograde signaling processes that emerge from DNA damage to cell cycle checkpoint control is
23 tempered by the fact that much of our information has been obtained to date, using rather high (most times supra-lethal) doses of agents, such as IR. In this chapter, we will examine the transcription factors activated after IR, with particular emphasis on doses required for their activation. For our discussion, ‘activation’ refers to conversion of an inert protein to an active form that can stimulate transcription of a gene. Expression of the new transcript or protein as a result of the ‘activation’ of these transactivation factors is an ‘induced’ gene product, such as the growth arrest and damage inducible (GADD) gene products (112) or x-ray–induced transcripts/proteins (XIPs) (91). We will focus on the activation processes of transcription factors following low, clinically relevant doses of
IR.
Signals from non-DNA targets
Much less is understood about IR-induced damage signaling from non-DNA targets that eventually cause transactivation of specific proteins (i.e., transcription factors) at clinically relevant doses of IR. Most of the studies attempting to elucidate these signaling events from non-DNA targets have utilized supra-lethal doses of IR and the triggering mechanisms have, in general, not been elucidated. Specifically, exposure of cells to IR results in a spectrum of lesions in cell membranes (e.g., lipid peroxidation).
Lipid peroxidation can result in the activation of PKC, MAPK, casein kinase, the ceramide pathway, JNK, ERK and other signal transduction pathways. In this way, damage from cellular plasma membranes is signaled inward to activate specific transcription factors. As stated above, these pathways have not been, in general, evaluated at low doses of IR, and the relevance of such signal transductions in the
24 Figure 1.3. DNA damage resulting from ionizing radiation exposure results in activation of signaling cascades that lead to either cell survival or apoptosis.
25 activation of specific transcription factors will be evaluated below for each known IR- stimulated transcription factor. Since IR causes simultaneous damage to DNA and non-
DNA targets, it can be a daunting task to evaluate the relative strength of DNA versus non-DNA targets for triggering a given signal transduction process. This, in turn, makes it difficult to determine the consequences of a particular activated signal transduction and/or transcription factor pathway in the overall response of the cell to IR. In particular, the use of high doses of IR to stimulate signal transduction, transcription factor activation, cell cycle checkpoint, or apoptotic responses in a more rapid timeframe (a rationale used by many investigators for the use of supra-lethal IR doses) appears to create many simultaneous (and at times conflicting or synergistic) signals that are clearly not applicable to clinically relevant lower doses of IR. These issues will be addressed in our discussion of known IR-activated transcription factors below.
Known IR-activated transcription factors
A review of the literature indicates a fairly select number of transcription factors activated by IR at any dose tested and in a variety of cell models. This list includes p53, nuclear factor-kappa-B (NF-κB), the retinoblastoma control proteins (RCPs), the early growth response 1 transcription factor (Egr-1, an Sp-1-like factor), the c-Fos/c-Jun AP-1 complex, and the octamer-binding protein-1 (Oct-1) transcription factor (Table 1.2). The transcription factors in Table 1 represent the known transcription factors activated by IR as found by a review of the current literature. In the sections to follow, we will discuss the data that demonstrates the IR activation of these transcription factors. We will then explore whether these transcription factors are activated at low clinically relevant doses
26 of IR and discuss their proposed functional significance in IR responses in normal or tumor cells.
IR stimulation of p53 transcriptional responses
The p53 tumor suppressor gene is mutated in ~50% of all human tumors and has been extensively reviewed (113-116). This review will specifically focus on the activation and functions of p53 by IR. Most of the studies investigating these functions have been done at high doses of IR (>4 Gy), while the role of p53 in low-dose responses still remains to be clarified. The p53 protein is tightly regulated and remains at low levels in unstressed cells (117), but is rapidly stabilized by various types of cellular stresses, including ionizing (IR) and ultraviolet (UV) radiations, hypoxia, stabilization/destabilization of microtubules, and ribonucleotide depletion (115, 118-120).
In response to DNA damage, p53 is rapidly stabilized through post-translational modifications that may include phosphorylation, acetylation, sumoylation, glycosylation and ribosylation. In general, IR is believed to activate (stabilize) p53 mainly through phosphorylation by the ATM kinase (113, 121, 122).
p53 plays a critical role in maintaining genomic integrity after cellular stress by acting as a transcription factor for a number of downstream genes that may mediate either cell cycle arrest or apoptosis, thereby preventing propagation of damaged DNA. The response to p53 stabilization is cell-type dependent, as well as damage dependent. For example, epithelial cells tend to undergo growth arrest after IR exposure, whereas lymphoid cells tend to undergo p53-dependent apoptosis (123). The signals that
27 Table 1.2
IR-inducible transcription factors.
Induction Transcription IR Dose Cell Type Used Time Reference Factor Range (Gy) Course (h) lymphocytic, p53 epithelial cancer 0.2-20 2-4 (124, 125) cells lymphocytic, NF-κB epithelial cancer 0.5-20 0.5-2 (126) (p50/p65) cells melanoma, Sp1 (RCPs) squamous 0.1-10 2-10 (127, 128) cell carcinoma lymphocytic, Egr-1 epithelial cancer 4-20 0.3-4 (129) cells lymphocytic, AP-1 epithelial cancer 2-20 1-5 (130, 131) cells, keratinocytes epithelial cancer Oct-1/NF-Y 5-20 4-8 (132) cells, keratinocytes
28 determine whether a cell undergoes cell cycle arrest or p53-dependent apoptosis after
DNA damage still remain to be elucidated.
The p53 protein consists of four major domains, a transactivation domain at the
N-terminus, a proline-rich domain, a DNA-binding domain and a carboxy-terminal tetramerization domain (133, 134). The transactivation domain contains the MDM2 binding site (135), as well as binding sites for members of the transcription machinery.
The proline-rich domain is important for efficient growth arrest after damage (136). p53 sequence-specific DNA binding occurs through the core DNA-binding domain (137).
After stress-induced stabilization, p53 forms homotetramers that bind to two copies of a ten-base pair (bp) nucleotide sequence (5’-PuPuPuC(A/T)(T/A)GpyPyPy-3’) divided by a 0-13 bp spacer (138). The importance of p53 sequence-specific DNA binding is emphasized by the fact that most mutations found in human tumors within the p53 gene occur at “hot spots” within this DNA-binding domain (139). These mutations result in a dominant-negative form of the protein that can heterodimerize with wild-type p53 protein and disrupt its DNA binding, and thus inhibit downstream gene activation. The C- terminal tetramerization domain is (as its name implies) responsible for tetramer formation, as well as for regulation of DNA binding by the core domain (136).
As mentioned above, p53 has a very short half-life in unstressed cells, and thus cellular protein levels are strictly regulated at low levels by a variety of mechanisms. The stability and activity of p53 can be influenced in several ways: (1) through ubiquitin- mediated proteolytic degradation; (2) through subcellular localization of the p53 protein; and (3) through allosteric regulation of the core DNA-binding domain by the C-terminal regulatory domain. In the first instance, the Mdm2 protein, which acts as an E3 ubiquitin
29 ligase for p53 (140-142), binds to the N-terminus of p53 and results in its rapid degradation through the proteasome pathway. Mdm2 also blocks p53 activity by binding to the region of the peptide required for transcriptional activation of downstream genes. p53 is a transcriptional activator of the mdm2 gene and thus generates a negative feedback loop that tightly controls the amount of p53 present within the cell.
Phosphorylation of Ser15 and Ser20 within the Mdm2-binding domain of the p53 protein lowers the affinity of p53 for Mdm2 and inhibits degradation. Ser6, Ser9, Thr18, Ser33,
Ser37, and Ser46 are also phosphorylated within this domain and may enhance p53 stability as well as influence acetylation of the C-terminus (discussed below) (113, 122).
Additionally, phosphorylation of Mdm2 can also disrupt the interaction of these two proteins and result in stabilization of p53 (143).
For p53 to bind DNA and act as a transcription factor, the stabilized protein must be localized to the nucleus. In this fashion, activation of p53 can be regulated through its subcellular localization. p53 contains a nuclear localization sequence and a nuclear export sequence within its carboxy-terminal domain. Moll et al. (144, 145) demonstrated that in certain breast cancer and undifferentiated neuroblastoma cell lines, p53 is inactivated by sequestration in the cytoplasm. Mdm2 may also act to export p53 out of the nucleus (146), in addition to its ability to mediate p53 degradation.
The carboxy terminus of the p53 protein acts as an allosteric regulator of sequence-specific DNA binding. This was demonstrated initially by Hupp et al., (147) using a bacterially expressed protein. Recombinant bacterial p53 bound poorly to DNA, and binding could be enhanced by the addition of antibodies specific to the C-terminal region of the protein. Phosphorylation of Ser315 and Ser392 within this domain also
30 enhance sequence-specific DNA–binding. Dephosphorylation of Ser376 of p53 after IR allows the association of 14-3-3 proteins with the C terminus of the protein (148).
Stavridi et al. (149) demonstrate that this interaction is required for p53 to activate the
downstream gene, p21/waf1/cip1, and for the G1 cell cycle checkpoint arrest response.
Interestingly, this dephosphorylation event seems to be ATM-dependent, possibly by a phosphatase that is activated by ATM after IR (148). p53 also binds nonspecifically to short single-strand DNA fragments through its carboxy domain, and this binding may enhance sequence-specific DNA binding, thus providing a potential role for p53 in DNA repair after damage. However, the significance of p53 associating with single stranded
DNA is not known.
In addition to phosphorylation, the C-terminal domain of p53 can also be acetylated and sumolated in response to DNA damage. Acetylation and sumoylation both result in an increase in the transactivation ability of p53 and may account for this finding. In vivo, IR induces the acetylation of p53 at Lys320 by PCAF and Lys382 by
CBP/p300. Acetylation at these sites is dependent on N-terminal phosphorylation at Ser15 and to a lesser extent on phosphorylation at Ser6, Ser9 and Thr18 (122, 150). All of these phosphorylation events are ATM-dependent, although only Ser15 has been shown to be phosphorylated directly by ATM. Sumoylation occurs at Lys386 after DNA damage
(151). Sumoylation refers to the covalent attachment of a small ubiquitin-like molecule
(SUMO-1) to Lys residues, but in contrast to ubiquitination, does not result in proteolytic degradation. The significance of sumoylation after IR still remains to be elucidated.
31 p53 stabilization by IR
The signals upstream of p53 stabilization and activation after IR exposure most likely originate from DNA DSBs. This is supported by the fact that the kinases implicated in the phosphorylation of p53 are also implicated in DSB repair. These include two kinases that belong to the PI-3 kinase family, DNA-PK and ATM (see above), and indirectly, the checkpoint kinase 2 (Chk2). Ser15 of p53 was identified as a substrate for DNA-PK in vitro (152). Since DNA-PK is required for DSB repair in mammalian cells after IR exposure and since this residue falls within the Mdm2-binding domain of the protein, this was an attractive model of p53 stabilization after IR.
However, several recent reports have shown that Ser15 is phosphorylated in DNA-PK deficient cells, and that p53 accumulation in these cells is capable of generating cell cycle arrest or apoptosis after IR (153-155). ATM is another kinase that has been shown to phosphorylate Ser15 in response to DNA damage induced by IR in vitro (156, 157). In contrast to DNA-PK–deficient cells, AT lymphoblasts that lack the ATM gene have a decreased ability to generate p53-dependent cell cycle arrest or apoptosis after IR, suggesting an important role for ATM in generating these responses in vivo.
Additionally, ATM has been implicated in phosphorylating Ser9 and Ser46 directly and
Ser20 indirectly through Chk2 (158, 159). Interestingly, ATM has also been implicated as the kinase that phosphorylates Mdm2, thus disrupting the p53-Mdm2 interaction by phosphorylation of both of these proteins.
32 p53 target genes
As mentioned previously, the downstream target genes of p53 can be grouped generally into those required for growth arrest and those required for p53-dependent apoptosis (136). p21/waf1/cip1, 14-3-3σ and GADD45 are three genes that are activated by p53 after IR and that are involved in growth arrest. p53 directly transactivates the p21/waf1/cip1 gene, which is an inhibitor of cyclin-dependent kinases (CDKs). By
inhibiting CDKs, p21 blocks the cell cycle at the G1-S transition as well as the G2-mitosis transition. Additionally, p53 can help to block cells in the G2 transition through its activation of the 14-3-3σ gene, which sequesters proteins required for entry into mitosis in the cytoplasm. GADD45 interacts with the repair protein PCNA and inhibits the entry of cells into S phase. It is important to note that the studies identifying these genes as targets of p53 transactivation were done with high doses of IR. It has been shown that p21/waf1/cip1 and GADD45 are induced after low-dose IR exposure (>2 cGy) (160), but the exact role of p53 in cellular responses to low doses of IR are not known.
The p53-activated genes involved in IR-induced apoptosis are less clear and appear to vary with cell type. Bax was the first apoptotic gene identified that was transactivated by p53 and has been implicated in IR-induced apoptosis in testicular germ cell lines (161, 162). A recent paper by Fei et al. (163) describes the tissue-specific induction of several p53-dependent apoptotic genes (KILLER/DR5, Bid, Puma, Noxa) after 5 Gy total body irradiation. Other groups have also investigated the transactivation of apoptotic genes after high dose IR (163-165), but little is known about the p53- dependent activation of these genes after clinically relevant doses.
33 IR stimulation of NF-κB responses
NF-κB is a transcription factor that exists as a latent form in association with the inhibitory protein IκB. It was first discovered as a nuclear factor that bound to the κB element present in the immunoglobulin kappa light chain gene in 1986 (166). It soon became apparent that it exists in most mammalian cell types as an inducible complex sequestered in the cytoplasm [reviewed by (167, 168)]. Many signaling pathways are known to cause degradation of IκB to release free NF-κB that then migrates into the nucleus and regulates induction of a wide variety of genes through decameric κB-binding sites. NF-κB target genes are known to play roles in a diverse array of cellular and physiological functions, including apoptosis, proliferation, cell adhesion, migration, and inflammatory and adaptive immune responses (reviewed in (169-172). While most studies focusing on the activation mechanisms and functional significance of NF-κB involve the use of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNFα) and IL-1, NF-κB has also been known to be activated by IR since 1991 (126). Using electrophoretic mobility shift assays (EMSAs) to detect newly liberated nuclear NF-κB, the above group showed that an IR dose as low as 2 Gy was sufficient to activate NF-κB in the KG-1 human myeloid cell line, with the peak dose at 5 to 20 Gy and the peak time around 2 to 4 hours after irradiation (126). Like in many other NF-κB activation pathways known, a protein synthesis inhibitor did not prevent this activation pathway, indicating that de novo protein synthesis is not required. Since the above report, NF-κB activation by IR has also been observed in different cell systems (see below).
34 The IR doses required for maximal NF-κB activation vary greatly depending on the cell lines or systems analyzed. In the EBV-transformed 244B human lymphoblastoid cell line, as in the case for the activation of AP-1 (see below), 0.5 Gy of IR was shown to yield the maximal NF-κB activation (127, 173). In the U1-Mel human melanoma cell line, 3 to 4.5 Gy was optimum with higher doses giving reduced activation (127, 128).
By contrast, in human diploid fibroblasts, even doses as high as 20 Gy failed to activate
NF-κB (174). More complicating are the contrasting observations of NF-κB activation following whole body irradiation at different IR doses. Zhou et al. (175) reported that
NF-κB activation by IR (8.5 Gy) is tissue-specific and only detectable in bone marrow, lymph nodes, and spleen. In contrast, Li et al. (176) demonstrated the activation of NF-
κB in liver and kidney when mice were exposed to 20 Gy of IR. In cerebral cortex of
Sprague-Dawley rats, NF-κB activation was detectable at 5 Gy with a steady increase up to 30 Gy (177). It thus appears that different normal tissues possess differential IR sensitivity toward the activation of NF-κB in vivo. However, it is presently unknown whether lower, more clinically relevant doses of IR (<2 Gy) would also yield activation of NF-κB in these normal tissues. Since NF-κB activation by IR can be readily observed in different human cancer cell lines derived from various tissue types in vitro (see below), but not normal counterparts (S. Miyamoto, unpublished observations), it is intriguing to consider the possibility that transformation or carcinogenic events may sensitize cells to
IR for activation of NF-κB. Ashburner et al. (174) found that immortalization of human fibroblasts by SV40 was correlated with acquisition of NF-κB activation capacity following IR treatment, further supporting this idea. Thus, it is possible, and even likely,
35 that cancer cells in vivo may display higher sensitivity to IR for activation of NF-κB, when compared to normal counterparts.
While activation of NF-κB can be affected by tissue types and transformed states, in those systems where NF-κB activation by IR can be readily observed distinct molecular components and signaling events have been implicated under different experimental settings. These include protein tyrosine kinases (PTKs), PKC, ROS, Ras,
ATM, DNA-PK, IκB kinase (IKK), and the ubiquitin-proteasome pathway (Figure 1.4).
Of particular interest with regard to the mechanism of activation of NF-κB by IR is the possibility that nuclear DNA damage may be directly involved in the activation of the cytoplasmic NF-κB/IκB complex, i.e., a novel nuclear-to-cytoplasmic, retrograde signaling pathway. Huang et al. (178) demonstrated multiple lines of evidence for the existence of such a signaling pathway induced by another anticancer agent, camptothecin.
IR is known to cause DSBs and subsequently activate nuclear kinases, such as ATM
[reviewed in (179)]. ATM was critical for NF-κB activation by IR at 20 Gy using an
ATM-/- mouse model, and the kinase inhibitor wortmannin prevented such activation
(180). However, the requirement of ATM in NF-κB activation seems to be different at different IR doses. Earlier work by Jung et al. (181, 182) showed that NF-κB activation in the SV40-immortalized AT5BIVA human fibroblast cell line was still observed in these ATM-deficient cells exposed at 6 Gy of IR. However, the same group reported that
ATM was required for Iκ B degradation, nuclear translocation of NF-κB and transcriptional activation by NF-κB in the same cell systems upon exposure to 20 Gy of
IR (183). Thus, the contribution of ATM is apparently dependent on the dose of IR in this cell system. Moreover, the general role of ATM has been challenged by Ashburner
36 et al, (174), since neither AT nor wild-type human diploid fibroblasts (not immortalized by SV40) activated NF-κB. Similarly, while another nuclear ATM-related kinase, DNA-
PK, has been implicated in the activation of NF-κB after 10 Gy exposure of HeLa cervical carcinoma and M059K glioma cell lines (184), liver and kidney tissues from
DNA-PKcs, Ku70 and Ku80 knockout mice displayed no NF-κB activation defects when exposed to IR at 20 Gy (185). In the latter report, c-ABL was also found dispensable for this activation pathway at this high dose of radiation. Moreover, there are also conflicting results regarding the role of the IκB kinase complex, phosphorylation and degradation of IκB by the ubiquitin-proteasome pathway to release active NF-κB when cells are exposed to different doses of IR in different cell systems (185-190). While direct phosphorylation of IκB by both ATM and DNA-PK has been shown in vitro (182-
184), whether such a direct phosphorylation is indeed induced by IR in intact cells has not been established. Thus, it is presently unclear whether nuclear DSBs are indeed required for activation of NF-κB by IR, especially at clinically relevant doses.
A previous study mentioned above (126) implicated the involvement of PKC based on the effect seen with H7 inhibitor and down regulation of PKC by prolonged
TPA treatments. Using the human Ramos B cell line exposed to IR doses of 10 to 40 Gy,
Uckun et al. (191) have implicated the involvement of PTKs and possibly PKC in the activation of NF-κB. However, by comparing the kinetics and magnitudes of NF-κB responses in primary tonsillar B cells exposed to TPA versus 15 Gy of IR, Wilson et al.
(192) concluded that simple PKC activation is unlikely to account for this activation process. Uckun et al. (191) also suggested that the generation of ROSs might be upstream of PTK activation. ROSs were previously proposed to represent a common
37 second messenger for NF-κB activation by distinct signaling agents (193). Consistently, antioxidants (e.g., N-acetylcysteine (NAC)) have been shown to prevent NF-κB activation by IR in several cell lines exposed to a dose range of IR (0.5-30 Gy) (194-196).
However, accumulating evidence indicates that the role of ROSs in NF- κB activation is also cell-type–restricted (reviewed by (197-200)), and NAC can directly inhibit 26S proteasome activity that is essential for degradation of IκB to release free NF-
κB (201). Moreover, the site of IκBα degradation induced by IR can also be distinct from those induced by cytokines, since it was suggested to occur at the plasma membrane, rather than in the cytosol, in U251 glioblastoma cell line exposed at 10 Gy (202). The same group also indicated the involvement of the Ras signaling pathway in NF-κB activation by IR through the use of a recombinant adenoviral system that expresses the anti-Ras single-chain antibody fragment (190). Thus, there is the possibility that the activation of NF-κB may occur at the level of the plasma membrane at high doses of IR exposure. The upstream regulators or how the Ras pathway connects with NF-κB activation following IR exposure is not well defined yet. It is also unclear whether lower doses (<2 Gy) of IR also employ the same activation mechanism to release NF-κB in the above or other cell systems.
Putting these published observations together, it is apparent that there is no consensus on the activation mechanism for IR regulation of NF-κB activity, especially at clinically relevant doses of IR. It is unclear whether IR-induced DSBs represent the signal initiating events or whether other stresses (e.g., ROSs) mediate this activation pathway. Different IR doses seem to invoke distinct signaling events to cause activation
38 Figure 1.4. IR stimulates the phosphorylation of IKK, which results in the degradation of IκB allowing for NF-κB to move into the nucleus and activate downstream genes.
39 of NF-κB. More complicating is the undetectable nature of NF-κB activation in many normal tissues after whole body irradiation, even at a relatively high dose of IR (up to 8.5
Gy). Many diploid human, normal cell types are also refractory to NF-κB activation following IR treatment in vitro, even though TNFα can efficiently activate NF-κB in these cells. These observations contrast with findings that many human cancer cell lines derived from different tissue types are permissive for activation of NF-κB by IR exposure in vitro. It is, however, important to note that NF-κB activation was measured by
EMSAs in most of these studies. Recent studies indicate that NF-κB activation, as measured by reporter gene activation, can take place without an apparent increase in the
NF-κB DNA-binding activity (203). Further in-depth analyses of signaling pathways and differential IR sensitivity of human normal versus cancerous cell types for activation of
NF-κB are, therefore, warranted.
What is the role of NF-κB activation in biological response to IR? An earlier study showed that induction of IL-6 by 5 Gy of IR exposure of FH109 human embryonic fibroblast cell line was dependent on the κB-binding site present in its promoter (126).
Others found a similar observation in PC-12 rat pheochromocytoma (204) and HeLa cell lines (205). IL-6 is critical for acute inflammatory reactions seen in IR-treated tissues.
TNFα, IL-1α, IL-1β, intercellular adhesion molecule-1 (ICAM-1) and E-selectin are also critical for inflammatory responses. Zhou et al. (206) found that induction of TNFα, IL-
1α, IL-1β and IL-6 in spleen, mesenteric lymph nodes and bone marrow of Balb/c mice following exposure to 8.5 Gy of IR was significantly reduced in mice knocked out for the p50 subunit of NF-κB. Studies also indicated that ICAM-1 induction by IR in HeLa,
HaCaT keratinocyte line, human umbilical vein endothelial cells (HUVECs) and
40 microvascular endothelial cells depends on NF-κB activation (195, 207). Similarly, E- selectin induction by IR (0.5-10 Gy) was also found to depend on activation of NF-κB in
HUVECs (208). All of these genes possess κB sites in their promoters (reviewed in
(171)). Thus, there is a consensus that the synthesis of inflammatory cytokines and molecules following IR exposure is coordinated by the activation of NF-κB in both in vitro and in vivo situations.
Another important role of NF-κB in biological response to IR is the modulation of radiosensitivity. An earlier study showed that the radiosensitive phenotype of SV40- immportalized AT5BIVA cells exposed to 6 Gy of IR was reversed by the expression of an N-terminally truncated I-κB that inhibited constitutive NF-κB activity present in these cells (181). These studies suggested that NF-κB promoted radiosensitivity. The similar radiosensitizing role of NF-κB has been observed in the human MRCA5CV1 fibroblast line exposed to 5 Gy (209) and U1-Mel cells exposed at the dose range of 5 to 15 Gy
(128). In contrast, Wang et al. (210) showed that when the HT1080 fibrosarcoma cell line was treated with IR, inhibition of NF-κB activation by the stable expression of the
“super-repressor” IκB mutant caused significant radiosensitization, as measured by colony forming ability assays at doses of 5 and 10 Gy (but not at 2.5 Gy). This latter study indicated that NF-κB activation caused radioresistance, rather than radiosensitization. This finding has also been confirmed in the LOVO colorectal cancer cell line in vitro and in tumor volume reduction of its xenograft in vivo (211). Similarly, the role of NF-κB in radioresistance was also seen in the A172, MO54 and T94 human glioma cell lines at 1 to 10 Gy (212, 213), the 3SB mouse lymphoma cell line exposed to
5 Gy (214), the UM-SCC-9 head and neck carcinoma cell line at a dose range of 1 to 12
41 Gy (215), the KB head and neck carcinoma cell line at 2 Gy (216), HeLa cells exposed to
2 Gy (217), and the HK18 human keratinocyte line at 2 to 12 Gy (218). These observations indicate that radiosensitization can be achieved by inhibiting NF-κB activation at the clinically relevant IR doses in many cancer cell types. Like the unresolved signaling mechanisms discussed above, the role of NF-κB in radioresistance is not straightforward and is heavily dependent on the specific cancer cell lines examined.
Several studies found that alteration of NF-κB activation did not affect radiosensitivity, either positively or negatively. These include the PC3 prostate cancer and HD-MyZ
Hodgkin’s lymphoma cell lines (219, 220), the U251 glioblastoma cell line (190, 202) and the RIE-1 rat intestinal epithelial cell line (221). Russell et al., (190, 202) further implicated the lack of NF-κB–dependent radioresistance in SF539 glioblastoma, SW620 and HT29 colon carcinoma, BXPC-3 pancreatic carcinoma, and C-39A normal fibroblast cell lines.
In conclusion, activation of NF-κB by IR can be observed in different cell systems at clinically relevant doses; however, there are also cell contexts in which it is not readily observed by EMSAs. In particular, activation of NF-κB in normal tissues versus primary tumors in vivo at low-dose IR needs further evaluation. Use of different assays to detect
NF-κB activation may also be required to fully appreciate the potential tissue specificity of this activation pathway in normal cells. In this context, a model developed by Carlsen et al. (222) to directly image NF-κB activation in vivo or development of similar assay systems could be most useful. While NF-κB activation by IR can be readily observed in a wide variety of cancer cell lines in vitro, the signal transduction mechanisms are complex and likely distinct at low versus high doses of radiation. There seems to be a
42 consensus regarding the role of NF-κB in mediating inflammatory responses to IR exposures, but its role in radiosensitization is highly dependent on the cancer cell line.
Importantly, it is entirely unclear whether these in vitro observations regarding the mechanisms and roles of NF-κB activation can be extrapolated into primary and metastatic tumors in cancer patients. Other studies employing 2D- versus 3D-culture systems have demonstrated that the sensitivity of specific cancer cell lines to radiation and anticancer drugs can be drastically altered by the presence of specific extracellular matrix components as well as cell polarity (223, 224). More importantly, activation of
NF-κB by etoposide and TNFα was also greatly modulated by the 3D architecture of the
T4-2 mammary tumor cell line (224). Since most of the aforementioned studies were performed under 2D conditions on plastic, it is critical to determine both the mechanisms and the roles of NF-κB activation by IR in modulation of radiosensitization under 3D culture and in vivo conditions. Even with these theoretical shortcomings, data to date are promising that NF-κB represents a novel anticancer target to enhance radiotherapy at clinically relevant doses. Such efforts have been spearheaded by multiple investigators, especially Baldwin and his colleagues (211, 225-227), to enhance not only radiotherapy but also chemotherapy. One of the major questions that remain to be resolved is: What are definable mechanism(s) that explain whether NF-κB plays a positive, negative or neutral role in modulation of radiosensitivity in specific cancer cell types in vivo?
Answers to this question will undoubtedly help target specific cancer types for enhancement of radiotherapy through NF-κB modulation, but will require analyses of
NF-κB activation mechanisms at clinically relevant doses of IR.
43 AP-1 transcription factor activation by IR
The ‘activating protein-1’ (AP-1) transcription factor was first identified as a protein responsible for regulating the expression of the human metallothionein promoter
(228, 229). Using AP-1 DNA-binding–site sequence-specific chromatography, several proteins were purified suggesting that the AP-1 transcription factor was a complex of more than one protein. Subsequently, analyses identified these proteins as members of the c-jun and c-fos protein families that can homo- or heterodimerize. The functional
AP-1 transcription activator complex was subsequently identified as containing the c-Jun,
JunB, JunD, as well as c-Fos, FosB, Fra-1 and Fra-2 proteins (230-235). Further studies identified additional binding partners (co-activators or co-repressors), such as ATF-2
(activating transcription factor-2) (236-240), Maf (241, 242), Nrf1 (238) and Nrl (242).
Jun proteins and their regulation
The v-jun oncogene was first isolated from the avian sarcoma virus-17 (jun is derived from ju-nana, the Japanese word for 17) (243). c-Jun and a number of Jun family members were isolated from mammalian cells based on their abilities to homo- and/or heterodimerize with themselves, Fos family members, or CREB/ATF, Maf and Nrl.
Various compositions of AP-1 result in differential DNA-binding affinities (e.g., c-Jun/c-
Fos > c-Jun/c-Jun > JunD/JunD > JunB/JunB), and some of these complexes can interfere with other AP-1 complexes (244, 245). It was shown, for example, that JunB may act as either a positive or negative regulator of transcription via binding to an AP-1 site (197,
246, 247). c-Jun protein is activated by phosphorylation of its N-terminal domain on residues Ser63 and Ser73. Substitution of these serine residues with alanine completely
44 inactivated Jun-dependent transcription (248). Analyses of c-Jun activation following UV irradiation led to the cloning of c-Jun N-terminal kinase (JNK), a member of the stress- activated protein kinase (SAPK) family (249).
Fos proteins and their regulation
The Fos protein family derived its name from the Finkel-Biskis-Jenkins murine osteosarcoma virus, from which v-Fos was isolated. c-Fos is a 55 kDa nuclear phosphoprotein (250). Fos family members are expressed predominantly in osteoblasts. c-Fos forms exclusive heterodimers with its binding partners, including the Jun family members ATF, Maf and Nrl, which are unable to form homodimers (242, 251). As in the case of c-jun, c-fos transcriptional activity is thought to be regulated by phosphorylation.
An enzyme called Fos kinase was isolated (252, 253), however, its role in the activation of AP-1 by IR has yet to be determined.
AP-1 characteristics
A common feature of proteins that form the AP-1 transcription factor complex is the presence of a leucine zipper that consists of heptad repeats of leucine residues aligned along one face of an alpha helix. Intercalation of leucine residues within the zippers of the two proteins and formation of a coiled-coil structure result in an AP-1 protein dimer.
Basic regions adjacent to the leucine zippers serve as the DNA-binding domain of the
AP-1 factor. The AP-1 binding site, also known as the TRE (12-O-tetradecanoylphorbol- b-acetate (TPA)-response element) (229), has a palindrome consensus sequence
TGA(C/G)TCA. The AP-1 site is similar to the cAMP response element (CRE):
45 TGACGTCA. Binding of the Jun family members to the CREs either alone or in complex with ATF/CREB proteins was reported (245, 254). Many genes have AP-1 site(s) in their promoters; however, these are mostly genes that are relevant to bone biology (e.g., collagenase) and that are responsive to AP-1 induction (255). The induction of IL-6 by IR (2 Gy, 6 h) in lymphocytic cancer cell lines was shown to be at least partially dependent on AP-1 activity (126, 256)
AP-1 activation after IR
Soon after AP-1 was discovered, elevations in the steady state levels of c-jun, junB and c-fos mRNA after IR were reported (131, 257, 258). c-fos mRNA was induced in syrian hamster embryo cells within 3 h after 0.75 Gy of x-rays or 0.9 Gy of gamma- rays (258). In contrast, no detectable c-fos mRNA induction was observed after treatment with high-LET fission-spectrum neutrons. In separate experiments, increases in c-jun, c- fos and junB steady-state mRNA levels in human HL-60 human promyelocytic leukemia cells were not detected at IR doses below 5 Gy (131). fosB and junD were also shown to increase their mRNA steady-state levels after ≥5 Gy of IR in HL-60 cells (259).
Curiously, the majority of studies of induction of AP-1 components by IR have been performed on cells not related to bone tissue, where these proteins, particularly c-Fos, are supposed to have physiological significance. Furthermore, the doses of IR used were much higher than those commonly used in radiotherapy. After supra-lethal doses of IR, increases in c-jun and c-fos mRNA levels correlated with a number of detectable intracellular processes, such as transient down regulation of cdc2, cyclin A, cyclin B, and cdc25 genes (260), induction of interleukin-1 (261), and apoptotic DNA fragmentation
46 (262). However, no direct connection between these events was shown, and no transcriptional activation of AP-1 nor direct increases of c-Fos, c-Jun or AP-1 proteins were determined.
Responses of AP-1 components to the lower, clinically relevant doses of IR (0.25-
2 Gy) and induction of AP-1 activity as a transcription factor were later shown in
Epstein-Barr virus- transformed human lymphoblastoid 244B cells. Induction of c-fos mRNA, after as low as 0.25 Gy, and c-jun, after 0.5 Gy, was observed, and levels of these mRNAs peaked at 1 h, but decreased 4 h after IR treatment (263). Subsequent studies of the c-jun promoter region, using a chloramphenicol acetyl transferase (CAT) reporter, revealed that AP-1 and CCAAT DNA elements were required for induction of this gene by high-dose IR exposures (264). These data suggested that a positive feedback loop was possible in c-jun activation following supra-lethal doses of IR. The requirement of AP-1 for the induction of jun gene was further confirmed in hypoxic HeLa cells (265). These data suggested the activation of a pre-existing AP-1 complex by IR via a post- translational mechanism(s). Indeed, the purification and cloning of JNK led to the elucidation of the c-Jun phosphorylation mechanism and AP-1 induction following various stresses, including IR (248, 249). Studies linking JNK activation and increased
AP-1 activity after low doses of IR remain to be performed in detail.
Subsequent studies have suggested that induction of c-Jun was dependent on PKC activation. This conclusion is based on evidence that depletion of PKC by pretreatment with TPA inhibited post-IR induction of the transcription of AP-1 components c-jun and c-fos (266). Also, TPA was shown to induce JNK activity (267). A similar dependence on PKC was demonstrated for c-fos gene induction after IR (268). New evidence derived
47 from the study of ataxia telangiectasia (AT) cells indicated the role of ATM in post-IR c-
Jun phosphorylation and activation (269). JNK is phosphorylated and activated by the
SAPK/ERK kinase (SEK1) pathway, also known as MKK4. MKK4 is, in turn, phosphorylated by MEKK1. Activation of MEKK1 after stress could be the result of activation of p21-activated kinase (PAK) via the Rho subfamily of small GTPases, in particular Rac1 and Cdc42. In fact, dominant-negative mutants of Rac1 or Cdc42 inhibited JNK activation stimulated by growth factors [reviewed in (270)]. It is not clear, however, whether these small GTPases participate in the IR induction of c-Jun phosphorylation. It is also yet to be determined if AP-1 activation and induction of c-fos or c-jun transcription via high-dose IR stimulation of JNK activity occurs after low, clinically relevant doses of IR. To date, nearly all of the studies concerned with elucidation of the signal transduction mechanisms of activation of the AP-1 transcription factor have used supra-lethal doses of IR.
Even though activation of AP-1 after IR has been described, it is not clear that activation of this transcription factor mediates downstream genes that have functional consequences for cellular responses to IR. Among the genes that have been clearly shown to be activated by AP-1 after IR are various cytokines, including TGF-ß1 (271), vascular endothelial growth factor VEGF (272), and IL-6 (205). Interestingly, none of these genes have been found to be responsive to AP-1 activation alone. In the case of
TGF-ß, it was shown that AP-1 activation alone was, in fact, not sufficient for the induction of the gene transcript (273). Likewise, AP-1 acts synergistically with NF-κB
(205) to induce IL-6 transcription. Clearly, cells have evolved complex mechanisms for gene induction after IR, as well as other stresses. This fact is highlighted by the failure of
48 multiple researchers to find a majority of known AP-1 transcriptionally dependent downstream genes induced in cells under conditions known to activate AP-1 after IR.
Additionally, it should also be noted that AP-1 activation was not found in all cells after
IR exposure (127), indicating cell specificity in this IR-responsive transcription factor and probably in JNK/SAPK/MEKK1 activation.
Sp1 activation after low doses of IR
Sp1 was the first identified member of a family of transcription factors composed of sixteen different proteins that bind to similar sequences in the promoter regions of human genes (274). Sp1 is ubiquitously expressed in mammalian cells and binds with high affinity to GC-rich sequences called GC-boxes, and with a lower affinity to CACC- elements called GT-boxes. Sp1 is thought to play a role in various cellular processes, including cell cycle regulation, chromatin remodeling and maintenance/propagation of methylation-free CpG islands. The importance of Sp1-mediated transcription is underscored by the fact that Sp1 null mice are embryonic lethal by day 10. This family of transcription factors includes the Sp family members (Sp1, Sp2, Sp3 and Sp4) and the kruppel-like family members. Sp1 and Sp3 have additional isoforms as a result of trans- splicing between pre-mRNAs or alternative translation initiation, respectively, that add to the complexity of this family of transcription factors. Additionally, Sp3 can act to repress transcription of Sp1-dependent promoters by competing with Sp1 for the DNA-binding site.
Sp1 and Sp3 are retinoblastoma (Rb) control proteins (RCPs) that bind to promoter regions, known as retinoblastoma control elements (RCEs). RCPs are regulated by the Rb protein and control induction of several immediate early growth-response
49 genes (IEGs) after cell stress (i.e. c-fos, c-jun, c-myc and TGF-β1). Two reports have shown the inducibility of Sp1 DNA binding after high doses of IR (>4 Gy). Activation of
Sp1 DNA binding after lower doses of IR has not been investigated in detail to date, although changes in Sp1 post-translational modification were observed after as little as 2 cGy IR (94). Yang et al. (128) demonstrated increased Sp1-related, RCP DNA binding to an RCE in radioresistant human malignant melanoma (U1-Mel) cells after 4.5 Gy, which correlated with an increased expression of IEGs; although low doses of IR were, in general, not used in this study, exposure of U1-Mel cells to this dose of IR resulted in only 60% lethality and greater than 50% survival within the 4-h potentially lethal damage recovery period allowed. Meighan-Mantha et al. (132) demonstrated a 5-fold increase in
Sp1-binding activity in head and neck squamous cell carcinoma after 15 Gy. Further research needs to be done to explore the possible role(s) of Sp1 and the complex RCP binding to RCEs within specific genes during cell death or survival responses after low- dose IR exposures.
IR-activation of the Egr-1 (Sp1-related) transcription factor.
The early growth response 1 gene (Egr-1, a.k.a., NFGI-A, Krox-24, TIS-8, or zif/268) product is unique among transcription factors, since both the transcription factor
(129, 275) and the gene transcript (129, 276) are both activated and induced, respectively, by supra-lethal doses of IR. The Egr-1 gene encodes a nuclear phosphoprotein with a cysteine-histidine zinc finger motif that is homologous with the Wilm’s tumor susceptibility gene and binds to the Sp1-like DNA element 5’-CGCCCCCGC-3’ (277,
278). Egr-1 belongs to a class of transcription factors that includes Sp1 (278), TFIIIA
50 (279), and SW15 (280). Along with the reported IR inducibility of Egr-1, the gene is also reported to be induced by various mitogenic stimuli, all believed to be the result of v-Src and v-Fps protein kinase activities (281, 282).
To date, the minimum dose of IR required to induce Egr-1 transcription or activation of transcription factor binding is 5 Gy, and has been reported in a variety of cancer cell lines, including human HL-525 myeloid leukemia, U87 malignant glioma, and
A375-C6 melanoma as well as in the AsPc-1 human pancreatic tumor cells (129, 260,
283-285). Induction of Egr-1 was prevented by the free radical scavenger NAC (129), however, the transcription factors responsible for regulating the gene’s promoter at
proposed serum response elements (SREs) and/or CC(A/T)6GG sequences remain unexplored (129, 283, 284, 286). Only one study has investigated the potential functional significance of Egr-1 induction on radiosensitivity, specifically using the A375-C6 melanoma cell line (284). Using stable expression of an Egr-1 dominant-negative mutant, as well as transient expression of Egr-1 antisense, Egr-1 levels were diminished and the effects of these changes on radiosensitization in terms of ‘percent growth inhibition’ were monitored following IR (5 and 20 Gy doses were used). Rather modest effects of antisense and dominant-negative Egr-1 expression on radiosensitivity were reported. The results were interpreted to indicate that Egr-1 is required for the growth- inhibitory response of these melanoma cells to IR. Egr-1 knockout studies and the use of clinically relevant doses of IR using colony forming ability assays would be required for definitive proof of this hypothesis.
Considering that the Egr-1 promoter has been one of the few IR-inducible sequences selected for ‘proof of principle’ gene therapy (discussed below) for the
51 proposed delivery of the bystander death gene, TNF-α (192, 208, 287, 288), it is surprising that: (a) such little information is available about the signal transduction pathways that regulate Egr-1 induction in a variety of cell types; and (b) there is paucity of data to indicate that the Egr-1 promoter can be induced by doses of IR that would be considered relevant to clinical therapy. Analyses of the published literature revealed that only one study has examined the signaling pathways that may stimulate the Egr-1 promoter at somewhat reasonable doses of IR (~5 Gy) (286). In this study, the Erk1/2 and SAPK/JNK signal transduction pathways were implicated, based entirely on the use of inhibitors of these specific pathways. The role of PKC was also implicated (130, 289-
291), however, these studies utilized supra-lethal doses of IR to leukemic cells, and the reported results were not applicable to clinically relevant doses of IR. A further review of the literature indicates that not all studies have found Egr-1 induction in mammalian cells after IR, and that in some cells the induction or expression of Sp1 can interfere with specific regulatory elements in the Egr-1 promoter (275). In summary, stimulation of
Egr-1 is observed in human cancer cells, however, supra-lethal doses of IR are generally required for the observed changes in transcription factor activation and induction.
Oct-1 and NF-Y transcription factors
A number of recent papers have demonstrated activation of octomer binding protein 1 (Oct-1) and/or NF-Y transcription factors in response to stress (132, 292-294).
Most of the data reported to date have used agents other than IR, such as methylmethane sulfonate (MMS), camptothecin, etoposide, cisplatin, or ultraviolet radiation (UV). A significant induction of Oct-1 (5-fold), monitored by electrophoretic mobility assays
52 (EMSAs), was observed in PCI-04A head and neck squamous cell carcinoma cells within
4 h after 5 Gy (132). Similar, albeit significantly less, Oct-1 binding activity was also reported in PC-3 human prostate cancer, as well as MDA-MB-231 human breast cancer cells after 5 Gy. Activated Oct-1 binding was more rapid and prolonged (lasting 0.5 –
16 h post-treatment) using supra-lethal doses of most cytotoxic agents; for example, such responses were reported in PCI-04A cells after 20 Gy. Lower doses of IR have not been explored, and changes in promoter activity before vs. after IR, using an Oct-1-dependent promoter, remains to be performed.
As with many activated transcription factors, particularly after exposure of cells to supra-lethal doses of IR, it is unclear as to the exact function of activated Oct-1 and/or
NF-Y DNA-binding activity on gene expression. One study found simultaneous activation of Oct-1 and NF-YA DNA-binding activities were essential for the p53- independent induction of the GADD45 promoter after lethal doses of MMS (100 µg/ml) or UV (10 J/M2) in HCT116 human colon carcinoma cancer cells (295). Separate mutations within the two Oct-1–binding sites or the NF-YA CAAT box abrogated induction of the GADD45 promoter. Unfortunately, IR exposures were not examined in this study. In a study of the secretion of von Willebrand factor (VWF) after IR, activated
NF-Y DNA-binding activity to a CCAAT element within the –90 to +22 core promoter was found to be essential for this gene’s promoter activity after lethal doses of IR (294).
Since VWF is an important factor in thrombus formation, understanding the regulation of this secreted factor after IR may be very important for controlling IR-induced thrombus formation.
53 The activation of Oct-1 DNA binding has not been a universal property of IR- exposed cells. Sahjidak et al. (127) found that Oct-1 levels were not altered in radioresistant human melanoma cells before or after various doses of IR (2-10 Gy) compared with normal basal level DNA-binding activities using EMSAs and 50-bp oligomers containing the octamer-1–binding motif. Factors such as cell type specificity or specific growth conditions that may affect the activation of Oct-1 or NF-Y DNA- binding activities after IR in mammalian cells have yet to be determined. Analyses in vivo of VWF and Oct-1 or NF-Y activities may also be warranted.
Exploiting IR-inducible Promoters and Transcription factors for improved cancer therapy
Over the past ten years or more, an extensive effort by many laboratories has attempted to understand the cellular and molecular biology of stress induced by IR.
Unfortunately, most of these studies have focused on signal transduction events occurring after relatively high, non-clinically relevant doses of IR that subsequently activate transcription factors and corresponding downstream promoters (reviewed above, Table
1). The assumption has been that one can then extrapolate down to low doses of IR and assume that the same changes in signal transduction events, activation of transcription factors (as well as co-activators and co-repressors), stimulation of specific promoters, and induction of IR-inducible gene products occur at low, clinically relevant doses of IR. This strategy has been used in an attempt to exploit the Egr-1 promoter as described above
(290). To date, little success in this clever approach has been achieved. Overall, the accumulated literature on IR-inducible gene expression seems to indicate that a better
54 understanding of low-dose IR-inducible promoters of specific genes in solid tumors, and not the more radiosensitive lymphocytes, is in order. More research focusing on gene induction events occurring in solid tumors (or cells from solid tumors) after IR doses between 0.5-2 Gy is needed. Extrapolation from high (many times supra-lethal) doses of
IR to clinically relevant exposures may often be misleading.
Examination of the literature indicates that two transcription factors, p53 and NF-
κB, are reproducibly responsive (120, 296-303) to clinically relevant (and sometimes lower) doses of IR. These data suggest that promoters regulated by these transcription factors (e.g., the p21- or 3X-κB–containing promoters) may be useful for IR-inducible expression in gene therapy regimens. For example, Ueda et al. (304) used the NF-
κB–responsive c-IAP2 promoter to drive the expression of the pro-apoptotic Bax gene to induce cell death at 2 Gy. More research is needed to explore IR-inducible RCPs and
RCE-directed promoters. Since these transcription factors are activated under conditions where cells survive and recover after IR, more extensive analyses of their induction and signal transduction pathways are warranted.
55 Chapter 2: Repression of IR-inducible clusterin expression by the p53 tumor
suppressor protein
This work was published in Cancer Biology and Therapy
Cancer Biol Ther. 2003 July 1; 2:4; 372-380
ABSTRACT
The clusterin (CLU) protein has been reported to have both cytoprotective and cytotoxic activities. Previous data from our lab suggest that the secretory form of CLU
(sCLU) is cytoprotective and induced after very low, nontoxic doses of ionizing radiation
(IR: >0.02 Gy), while a nuclear form is cytotoxic (95). Cells must presumably suppress sCLU to stimulate cell death, however, factors regulating the stress-inducible expression of sCLU have not been elucidated. Here we demonstrate that p53 can suppress sCLU induction responses. A variety of cytotoxic agents stimulated sCLU expression and DNA damage was sufficient but not necessary for induction. IR-stimulated CLU promoter activity, with concomitant increases in CLU mRNA and protein, showed that CLU induction was delayed with maximal expression observed 48-96 h post-treatment.
Expression of the human papillomavirus E6 protein in MCF-7 breast or RKO colon cancer cells enhanced basal sCLU levels. Isogenically matched HCT116 colon cancer cell lines that differed only in p53 or p21 status, confirmed a role for p53 in the transcriptional repression of sCLU. Loss of functional p53 in HCT116:p53-/- cells augmented sCLU de novo synthesis after IR exposure. Repression of sCLU protein
56 levels by p53 may be important for the cascade of p53-mediated events leading to cell death after IR or other cytotoxic agent exposure.
INTRODUCTION
Clusterin (CLU) is a sulfated glycoprotein implicated in many physiological and pathological processes, including tissue remodeling (21), complement inhibition (14,
305), lipid transport (9, 37), multiple sclerosis (306), atherosclerosis (307, 308) and
Alzheimer’s disease (39, 41, 309). Two different forms of the CLU protein exist: an 80 kDa secretory form composed of 40 kDa alpha and beta subunits and an ~55 kDa nuclear form. Our laboratory identified CLU as a x-ray inducible protein/transcript (xip8) (11).
We showed that a nuclear form of CLU (nCLU) associated with the DNA double strand break (DSB) repair protein, Ku70, and was a pro-death factor (35, 71). However, the secretory form of CLU (sCLU) did not associate with Ku70, and this form of CLU was induced by much lower, nontoxic doses of IR. In fact, sCLU was induced at ~0.02 Gy, a dose found to be growth-stimulatory and cytoprotective in many human cancer cells (71).
The regulation of sCLU remains poorly understood.
Elevated levels of sCLU protein and mRNA were noted in several different types of human malignancies (49, 57), and forced over-expression of sCLU in transformed cell lines resulted in an increased resistance to various chemotherapeutic agents (48, 62). In addition, abrogation of CLU mRNA expression following antisense expression lead to modest chemo- and IR-sensitizations in various cell lines (72-74, 310). These data support a cytoprotective role for sCLU.
57 The p53 tumor suppressor gene is mutated in over half of all human tumors (311).
Wild-type p53 protein is stabilized after cellular stress and acts as a transcription factor for various downstream genes, including Bax, p21 and GADD45, resulting in either cell cycle arrest or apoptosis (312-314). p53 can also act as a repressor of transcription, although exact mechanisms of transcriptional suppression still remain to be elucidated.
Examples of p53-repressed genes include presenillin (315), hsp70 (316), cyclins A(317) and B (318) and cdc2 (319).
Our laboratory identified CLU as a x-ray inducible protein/transcript (xip8) (11).
We showed that a nuclear form of CLU (nCLU) associated with the DNA double strand break (DSB) repair protein, Ku70 (35, 71). However, sCLU did not associate with Ku70, and this form of CLU was induced by much lower, nontoxic doses of IR. In fact, sCLU was induced at ~0.02 Gy, a dose found to be growth-stimulatory and cytoprotective in many human cancer cells (71).
Although regulation of CLU gene expression following estrogen and testosterone exposures has been investigated (64, 320), the regulatory control of sCLU synthesis after
IR or other cytotoxic agents has not been elucidated. We show that CLU mRNA and protein synthesis in human cells is induced after various cytotoxic stresses, including exposure to many anti-tumor agents. IR-induction studies of CLU promoter activity, CLU mRNA accumulation, and sCLU protein synthesis confirm that sCLU expression occurs in a delayed fashion, with initial IR-activation of the CLU promoter occurring 24 h post- exposure, and mRNA and protein levels maximally accumulating 48-96 h post-IR. The low levels of IR (>0.02 Gy) that induce sCLU and the dramatic accumulation of sCLU protein following taxol, TPA or thapsigargin (a sarcoplasmic reticulum Ca2+ -ATPase
58 (SERCA) pump inhibitor that causes dramatic alterations in intracellular calcium homeostasis) exposures, suggest that DNA damage may not be required for CLU gene expression in MCF-7 breast cancer cells. Expression of the human papillomavirus (HPV)
E6 protein, as well as isogenically matched cell lines that differ only in their p53 status, were used to demonstrate a role for p53 in the transcriptional repression of sCLU in unirradiated as well as IR-exposed cells. Loss of functional p53 results in elevated basal levels of sCLU in some cells, and augmented IR-induced gene expression in all cells examined. These data strongly suggest that sCLU mRNA production and protein synthesis are repressed by the tumor suppressor protein, p53.
EXPERIMENTAL PROCEDURES
Chemical Reagents
Camptothecin, etoposide, colcemid, nocodazole, taxol, mimosine, TPA, and thapsigargin were obtained from the Sigma Chemical Co (St. Louis, MO) and dissolved in either PBS or DMSO. Topotecan was generously provided by Glaxo SmithKline (Research Triangle
Park, NC). ß-Lapachone was prepared for us by Dr. William G. Bornmann (Synthetic
Preparatory Core Facility, Memorial Sloan Kettering, NY, NY). Taxotere was generously provided to us by Aventis Pharmaceuticals (Bridgewater, NJ).
Cell Culture
MCF-7:WS8 human breast cancer cells (MCF-7) were obtained from Dr. V. Craig Jordan
(Northwestern University; Evanston, IL). MCF-7 cells were transduced by retroviral transfer with a CMV-driven papillomavirus E6 vector by Dr. Jordan's lab, and
59 subsequently subcloned by our lab into cell lines with varying E6 expression. The E6-D
MCF-7 cell line showed no p53 expression, even after IR exposure. Human colorectal carcinoma HCT116 parental, p53-/-, and p21-/- cell lines were developed (321) and generously provided by Dr. Bert Vogelstein (Johns Hopkins University; Baltimore, MD).
These cell lines were confirmed by our laboratory to be null for p53 and p21, respectively, by western blot analyses. MCF-7, ZR-75-1, T47-D, BT474, MDA-MB-231 and MDA-MB-468 cell lines were grown in RPMI 1640 cell culture media supplemented
with 5% fetal bovine serum (FBS) at 37°C in a humidified incubator with a 5% CO2-95% air atmosphere as described (322). MCF-7:E6 cells were maintained in 0.4 mg/ml geneticin (Life Technologies; Carlsbad, CA). RKO:neo and E6-expressing RKO cell lines were obtained from the American Type Culture Collection, maintained in G418, and experiments performed in the absence of antibiotics. HCT116 and RKO cell lines were grown in DMEM supplemented with 10% FBS at 37°C in a humidified incubator with a
5 10 % CO2-90% air atmosphere. All experiments were initiated by seeding 5 x 10 log- phase cells per 10-cm2 tissue culture dish in the appropriate medium in the absence of any antibiotics (e.g., geneticin). All cell lines were free from mycoplasma contamination.
IR and Chemical Treatments
Cells were irradiated as described (11). Briefly, cells were irradiated with 137Cs gamma rays at a dose rate of 0.87-0.92 Gy/min, using a Shepard Mark Irradiator. Untreated cells were mock-irradiated as described (11). MCF-7 cells were treated with topotecan, camptothecin, mimosine, colcemid, nocodazole, TPA, thapsigargin, taxotere, taxol or etoposide using drug exposures at the indicated doses as described in Table 2. Cells were
60 treated with ultraviolet radiation as described (91). ß-Lapachone and hypoxic exposures
(323) of log-phase MCF-7 cells were performed as indicated in Table 2.
Northern Blot Analyses
Total RNA was extracted from control or irradiated MCF-7 or HCT116 cells as indicated using Trizol (Life Technologies; Carlsbad, CA) as per the manufacturer’s instructions.
Total RNA (10 - 20 µg) was separated on a denaturing formaldehyde gel, transferred to a
Hybond membrane (Amersham Pharmacia; Sunnyvale, CA), and probed with 32P-labeled full-length CLU or 36B4 cDNAs as described (91); 36B4 levels are not affected by cell stress, or cell cycle status (91). Corresponding transcript signals were quantified using
ImageQuant software version 4.1 (Molecular Dynamics; Sunnyvale, CA) on a Molecular
Dynamics phosphoimager. CLU mRNA levels were normalized to untreated control levels, and to 36B4 mRNA levels for X-fold induction calculations as described (91).
Luciferase Assays
All luciferase assays were performed using the Luciferase Assay System (Promega;
Madison, WI). MCF-7 cells were stably transfected with a 1403 bp fragment of the human CLU promoter in a luciferase reporter plasmid using a standard liposome transfection protocol (Effectene, Qiagen, Valencia, CA). The plasmid was a generous gift from Dr. Martin Tenniswood (University of Notre Dame; Notre Dame, IN). These cells (MCF-7:1403 cells) were seeded in 6-well plates at approximately 50% confluency.
Cells were irradiated at the indicated dose(s) and harvested at various times in 1X
61 reporter lysis buffer (Promega; Madison, WI). Each dose/time point was completed in triplicate and a Student’s T-Test was performed to determine statistical significance.
Western Blot Analyses
Whole cell extracts from control or irradiated cells were extracted in RIPA buffer (0.1%
SDS, 0.5% deoxycholate, 1% NP-40, 150 mM NaCl, 50 mM Tris pH 8.0) and separated on a 10% gel by SDS-PAGE western blot analyses as described (35). Proteins were transferred to Immobilon-P (Millipore; Bedford, PA) and probed with the B-5 human sCLU monoclonal antibody, the DO-1 human p53 monoclonal antibody, and the human
C-19 Ku70 polyclonal antibody. All antibodies were obtained from Santa Cruz and used as per manufacturer’s instructions. Ku70 was used as a control for equal loading of protein, since its levels remain unaltered after IR or cell cycle status under the time-frame of our experiments. Western blots shown are representative of experiments performed at least three times. For sCLU protein analyses in cells before and after IR, we routinely use the 60 kDa form, since all cell lines produce this 60 kDa protein, which is a precursor to the mature glycosylated α- and β- ~40 kDa polypeptides of sCLU.
Cell Cycle Analyses
HCT116 parental, p53-/- or p21-/- cells were synchronized by allowing them to grow to
100% confluence on 10-cm2 tissue culture dishes as described (11, 324). Cells were then
maintained for 48 h in serum-free medium to maximize G0-G1 arrest. Cells were released from the dual confluence and serum-free cell cycle arrest by trypsinization (using 0.05% trypsyn with 0.53 mM EDTA) and replated at 1:8-1:10 dilution in DMEM containing
62 10% FBS under conditions described above. For IR treatments, cells were exposed to 10
Gy, 10 h after release from the cell cycle. Concurrent flow cytometric and western blot analyses were performed as indicated. At various times after mock- or IR-exposures, cells were dissociated by scraping into 1X PBS, collected by centrifugation (500 x g), fixed in
90% ethanol, and stored at –20 0C until analyzed. Cells were then stained with 33 mg/ml propidium iodide (PI) (Sigma; St. Louis, MO), 1.0 mg/ml RNase A (Sigma), and 0.2%
NP-40 (Calbiochem; La Jolla, CA) at 4 0C overnight. Stained nuclei were then analyzed for DNA content by PI fluorescence using a Coulter Epics XL (Beckman Coulter
Electronics; Miami, Fl) flow cytometer. Data were analyzed using ModFit LT, version
2.0 software (Verify Software House; Topsham, ME). Western blot analyses were completed simultaneously with flow cytometry, and results shown represent experiments performed at least three times.
RESULTS sCLU is transcriptionally upregulated after IR.
Our laboratory previously showed that CLU was an x-ray-induced protein (xip8) (91), however, regulation of the CLU gene was not elucidated. To further characterize induction of sCLU after IR exposure, northern blots were used to determine if sCLU protein accumulation in log-phase MCF-7 human breast cancer cells after IR exposure was due to increased transcription, or a result of protein stabilization (Figures 2.1A,B).
Log-phase MCF-7 cells were mock-irradiated or exposed to 10 Gy and harvested at various times post-IR to analyze the temporal kinetics of CLU gene expression. Maximal induction of CLU mRNA (7- to 10-fold) over untreated cells occurred 72 to 96 h after 10
63 Gy (Figure 2.1A). Induction of the CLU transcript in MCF-7 cells after IR was confirmed using RNase protection assays (data not shown). IR dose-response experiments in log-phase MCF-7 cells were performed, and CLU mRNA accumulations
72 h after exposure were examined (Figure 2.1B), since maximal protein and mRNAs were noted at this time post-IR (Fig. 2.1A). As previously reported for sCLU protein induction at 72 h post-IR (35). CLU mRNA was induced 2-fold after as little as 2 cGy, with maximal induction of 22-fold in MCF-7 cells after 5 Gy (Figure 2.1B). Steady state
CLU mRNA accumulation corresponded well with previously described sCLU protein accumulation in MCF-7 cells after IR in terms of temporal and dose-response kinetics
(35).
To determine if IR-induced CLU transcriptional increases were due to de novo mRNA synthesis, or to decreases in mRNA degradation (i.e., via post-transcriptional modifications), we examined CLU promoter activity in time-course and dose-response studies after IR. For these experiments, we generated an MCF-7 cell line containing a stably integrated copy of a plasmid containing a 1403 bp fragment of the human CLU promoter directing expression of a downstream luciferase reporter as described in
'Experimental Procedures'. Transient transfections with the CLU reporter plasmid were problematic, since all transfection methods examined to date affected the regulation of the CLU promoter-luciferase construct in MCF-7 cells, as well as endogenous sCLU gene/protein expression (data not shown); induction of sCLU may be triggered by cell membrane insult (325). Dose-response (Figure 2.1C) and time-course (Figure 2.1D) assays of exogenous CLU promoter activation in MCF-7:1403 cells were performed to show that this clone behaved similarly to the endogenous CLU gene before and after IR
64
C A 1400 UT 10 Gy * 1200 *p<0.03 Time (h): 96 4 24 48 72 96 1000 800 1.8 kb CLU 600 * 400 * 0.7 kb 36B4 200 0 Fold 1 Induction: 1 1 2 8 7 8 4 24 48 72 96 4 24 48 72 96 Relative Luciferase Units UT 10 Gy D B 7.E+03 * X-irradiation (Gy) 6.E+03 *p<0.02 UT 0.02 0.05 0.1 0.25 0.5 1.0 2.5 5.0 10 5.E+03
4.E+03 * 1.8 kb CLU 3.E+03 * 2.E+03 0.7 kb 36B4 * 1.E+03 Fold 0.E+00 Induction: 1 2 2 4 5 7 11 13 22 7 1 0 0.5 2 5 10 Relative Luciferase Units X-irradiation (Gy)
65 Figure 2.1. sCLU is transcriptionally upregulated after IR exposure in MCF-7 human breast cancer cells. CLU mRNA levels were monitored in asynchronous
MCF-7 cells after 10 Gy by northern blot analyses and luciferase assays. In A, log- phase growing MCF-7 cells were irradiated with 10 Gy and 10 µg of total RNA was analyzed by northern blot analyses as described in Experimental Procedures. In B,
MCF-7 cells were irradiated with various doses of IR and total RNA was harvested
72 h after exposure. Total RNA (10 µg) was used for northern blot analyses. In C, time-course of sCLU induction after 10 Gy exposure was analyzed by luciferase assays in MCF-7 cells stably transfected with 1403 base pairs of the CLU promoter
(i.e., MCF-7 1403 cells) using the Luciferase Assay System (Promega). In D, an IR dose-response was performed on the MCF-7 1403 cells 72 h after IR exposure. Each dose/time point was performed in triplicate and a Student’s T-Test was performed to determine statistical significance. *p values denote IR compared to untreated
(UT) controls.
66 exposure. The CLU promoter was activated in a time- and dose-dependent manner similar to that previously shown for sCLU protein and mRNA (Figures 2.1A and B).
CLU promoter activity was stimulated by as low as 0.5 Gy.
sCLU is a stress protein induced by a variety of cytotoxic agents.
Table 2.1 lists various cytotoxic agents that induce sCLU protein expression in MCF-7 cells. These agents included ultraviolet radiation (UV), topoisomerase I and IIα poisons, microtubule stabilizers/destabilizers, as well as other agents that do not cause direct damage to DNA (e.g., TPA, thapsigargin). Treatment of MCF-7 cells with hypoxic conditions or various doses of β-lapachone (2-10 µM, 4h), a novel apoptotic drug that quickly depletes cellular NAD(P)H and ATP in NQO1-expressing MCF-7 cells (326), did not induce sCLU protein expression. These data suggest that damage to DNA may be sufficient, but is not required for sCLU induction. Alterations in calcium homeostasis
(indicated by thapsigargin induction of sCLU, Table 2.1) or ER stress responses may play a common role in triggering CLU gene induction.
Correlation of sCLU expression and loss of functional p53.
Various human breast, colon and prostate cancer cell lines with known mutations in p53 were examined for basal and IR-inducible sCLU levels as monitored by western blot analyses and described in 'Experimental Procedures'. With one exception, cells expressing mutant p53 exhibited increased basal levels of sCLU (Table 2.2). Mutant p53-expressing MDA-MB-231 cells appear to lack basal or IR-inducible CLU protein expression, and have no detectable CLU mRNA levels by Northern blot analyses (data
67 not shown). In contrast, cells expressing wild-type p53 expressed low or no detectable basal levels of sCLU (Table 2.2). With the exception of MCF-7 cells, we also noted that cells expressing wild-type p53 did not greatly induce sCLU expression after various doses of IR to the same extent as null or mutant p53-expressing cells.
The data in Table 2.2 indicate an inverse correlation between sCLU expression and expression of wild-type p53. To further elucidate the effect of p53 on sCLU expression, we compared vector alone-transfected parental MCF-7 cells to isogenically matched cells stably transfected with the HPV-16 E6 protein, as described in
‘Experimental Procedures’. The E6 protein binds to p53 and targets it for rapid degradation through the proteasome pathway, leaving these cells deficient (i.e., null) for p53 expression (327). Protein and RNA from log-phase MCF-7:parental and MCF-7:E6 cells were harvested at various times after exposure to 10 Gy. Consistent with the mRNA changes shown in Fig. 2.1, sCLU protein was induced in parental MCF-7 cells starting at
24 h, and levels peaked at 72 h post-10 Gy (Figure 2.2A). The 60 kDa band in Figure
2.2A is a precursor form of sCLU (psCLU) expressed in the ER and is cleaved at an α/β cleavage site resulting in two 40 kDa peptides that heterodimerize through five disulfide bonds to form mature 80 kDa sCLU protein. Western blots performed under denaturing conditions result in the appearance of a 40 kDa smeared band consisting of glycosylated
α- and β-peptides of sCLU. sCLU basal levels were higher in mock-irradiated MCF-
7:E6 cells compared to parental MCF-7 cells. As expected, p53 protein levels were not detected in MCF-7:E6 cells at various times before or after IR. Furthermore, induction of sCLU protein was enhanced in MCF-7:E6 cells after IR compared to levels in parental
68 Table2.1
Induction of sCLU protein expression in MCF-7 cells1 by various cytotoxic agents.
Agent Dose Range for Induction2 Level of Induction3 DNA Damaging Agents Ionizing Radiation (IR) 0.02 - 10 Gy +++ Ultraviolet Radiation (UV) 12 J/m2 +++ Topotecan 50 nM +++ Camptothecin 100 nM +++ Etoposide (VP-16) 15 µM +++ Non-DNA Damaging Agents Photodynamic Therapy (PDT)4 200 nM PC-4/200 mJ/cm2 ++ Colcemid 70 ng/ml ++ Nocodazole 150 ng/ml +++ Taxol 1 - 50 nM +++ Taxotere 1 - 10 nM +++ Mimosine 0.5 mM ++ TPA 100 nM + Thapsigargin 10 - 500 nM ++ Non-Inducing Agents β-Lapachone 2 - 10 mM NA 5 Hypoxia (<0.1% O2) NA
1Log phase MCF-7 cells were seeded at approximately 5 X 105 cells per 10 cm plate.
2Topotecan, camptothecin, TPA and thapsigargin were continuous treatments.
Cells were treated with colcemid, nocodazole and mimosine for 24 hours, washed with PBS and replated into fresh media. Cells were treated with taxol or taxotere for 4 hours, washed with PBS and replated into fresh media. Cells were treated with VP-16 for 1 hour. Protein was harvested at least 48 hours after drug addition/irradiation.
3Level of induction of sCLU protein as compared to sCLU protein derived from 10
Gy irradiated MCF-7 cells.
69 4Photosensitizing drug used was Phthalocyanine 4 (PC-4). Induction of sCLU protein was only seen after addition of drug and light exposure. No induction was observed with light alone or PC-4 alone.
5Hypoxia was induced as previously described (323).
70 MCF-7 cells after 10 Gy (Figure 2.2A). Since MCF-7:E6 cells have a higher basal level of sCLU, IR-induction of sCLU in these cells was more difficult to quantify, and
Northern
HPV-16 E6-expressing MCF-7 cells have high basal levels of sCLU. blot analyses indicate this fact (see below). Finally, we noted a similar dramatic increase of sCLU in the medium of IR-treated MCF-7 cells, and a significantly higher basal level of sCLU in the medium of MCF-7:E6 compared to MCF-7:neo vector alone parental cells (Klokov et al., unpublished data).
Northern blot analyses confirmed that basal CLU mRNA levels were 3-fold higher in mock-treated MCF-7:E6 cells compared to MCF-7:parental cells (Figure 2.2B).
Furthermore, CLU mRNA levels were only modestly induced in MCF-7:E6 cells (~3-fold from 4h to 96 h post-IR), compared to IR-treated vector alone MCF-7:parental cells, in which a 10-fold increase in CLU mRNA level was noted (Figure 2.2B).
RKO:neo and RKO cells stably expressing the E6 protein (RKO:E6) were mock- irradiated or treated with 5 Gy. Protein was harvested 72 h after exposure and analyzed by western blotting (Figure 2.2C). As with many other cells examined, RKO cells showed low levels of the mature 40 kDa glycosylated form of sCLU, presumably because this protein is secreted from the cell. Wild-type p53-expressing RKO:neo cells expressed low basal levels of sCLU protein, with a measurable IR-inducible expression of the 60
71 Table 2.2
Effect of p53 status on sCLU basal and IR-inducible expression.
IR Cell Line p53 Status Basal1 Inducibility2 RNA3 Breast cancer cell lines MCF-7:parental wild-type (wt) low yes + MCF-7:E6D wt (no expression) high yes + ZR-75-1 wt low no + T47-D mutant (194) high no + BT474 mutant (275) high no + MDA-MB-231 mutant (280) ND ND ND MDA-MB-468 mutant (273) high no + Colon cancer cell lines HCT116:parental wt low minimal + HCT116:p21 -/- wt low minimal + HCT116:p53 -/- null low yes + RKO:neo wt low minimal NP RKO:E6 wt (no expression) high yes NP Prostate cancer cell lines LNCaP wt low minimal NP DU-145 mutant (275) high minimal NP
1Basal levels were determined as compared to log-phase growing untreated MCF-7 parental cells.
2Log-phase growing cells were treated with 10 Gy IR and protein was harvested 48 hours after exposure. MCF-7 parental cells were used as the standard for “high” IR inducibility.
3RNA status was determined by RT-PCR using primers designed to full length CLU
DNA.
ND, not detected; NP, not performed
72 kDa sCLU protein form after IR. In contrast, RKO:E6 cells expressed high basal levels of the 60 kDa sCLU protein, similar to that seen in MCF-7:E6 cells. As in MCF-7 cells, sCLU induction was more dramatic in RKO:E6 cells compared to RKO:neo cells, suggesting that loss of p53 function relieves IR-induction responses of sCLU.
Somatic deletion of p53 in HCT116 colon cancer cells results in greater IR-inducible sCLU levels.
Since E6 expression may have additional unknown ‘gain of function’ properties, and to confirm the ability of p53 to repress sCLU expression after IR, we used isogenically matched human HCT116 colon cancer cell lines that differed only in their p53 or p21 status. As with RKO cells, we were not able to observe intracellular mature 40 kDa sCLU levels in HCT116 cells and IR-induced sCLU induction responses were monitored via the ~60 kDa sCLU precursor protein (psCLU). Protein and RNA from log-phase
HCT116:parental and HCT116:p53-/- cells were harvested at various times after exposure to 10 Gy. Western blot analyses showed that IR-treated HCT116:parental cells stabilized and accumulated p53 (i.e., expressed wild-type p53), but increases in steady state levels of sCLU were minimal to non-detectable (Figure 2.3A). In contrast, HCT116:p53-/- cells dramatically induced sCLU after 10 Gy. As found with RKO:E6 and MCF-7:E6 cells,
HCT116:p53-/- cells expressed higher basal levels of sCLU compared to the low levels
73 MCF-7:parental MCF-7:E6D A UT 10 Gy UT 10 Gy Time (h): 4 96 4 24 48 72 96 4 96 4 24 48 72 96 psCLU
sCLU
p53
Ku70
B UT 10 Gy UT 10 Gy Time (h): 4 4 24 48 72 96 4 4 24 48 72 96
1.8 kb CLU
0.7 kb 36B4
Fold Induction: 1 1 3 8 10 9 3 3 3 5 8 9
C RKO:neo RKO:E6 UT 5 Gy UT 5 Gy psCLU
p53
Ku70
Figure 2.2. sCLU basal levels are elevated in MCF-7 and RKO cells that over- express the HPV E6 protein. MCF-7:parental and MCF-7:E6 cells were exposed to
10 Gy and protein was harvested at various time points. In A, protein (100 µg) was loaded for each sample and separated by standard 10 % SDS-PAGE. Blots were probed for sCLU, p53 and Ku70 using western blot analyses as described in
Experimental Procedures. Ku70 was used as a loading standard as described. In B, total RNA (10 µg) was analyzed using standard northern blot techniques as
74 described in Fig. 2.1. Shown are representative blots from experiments performed at least three times. In C, RKO:neo and RKO:E6 cells were exposed to 5 Gy and protein was harvested at 72 h. Protein (100 µg) was loaded for each sample and separated by standard 10 % SDS-PAGE. Blots were probed for sCLU, p53 and
Ku70 using western blot analyses as described in Experimental Procedures.
PsCLU, presecretory 60 kDa clusterin form; sCLU, mature secretory 80 kDa clusterin form seen as 40 kDa glycosylated α - and β- polypeptides. Ku70 is a nonhomologous end joining protein that is not altered by cell cycle or cell stress changes.
75 noted in HCT116:parental cells. IR dose-response analyses of sCLU responses in
HCT116:p53-/- cells demonstrated induction of sCLU protein at doses as low as 1 Gy
(Figure 2.3B), a dose of IR that caused minimal loss of clonogenic survival (97).
Northern blot analyses confirmed induction (6- to 7-fold) of steady state CLU mRNA in
HCT116:p53-/- cells (Figure 2.3C), whereas p53+/+ HCT116:parental cells showed little or no induction of CLU mRNA at various times (up to 96 h) after 10 Gy.
To demonstrate that induction of sCLU in p53 null cells was specific for the absence of p53 and not a gene downstream from p53, we utilized HCT116 cells that were somatically knocked out for the p21 gene (i.e., HCT116:p21-/- cells) (321). As in p53+/+
HCT116:parental cells, sCLU protein and mRNA levels were only minimally induced in
HCT116:p21-/- cells after 10 Gy compared to mock-treated cells, as determined by western and northern blot analyses (Figure 2.4A, B).
76 A HCT116:parental HCT116:p53-/- UT 10 Gy UT 10 Gy Time (h): 4 96 4 24 48 72 96 4 96 4 24 48 72 96
sCLU
p53
Ku70
B X-irradiation (Gy) UT 1.0 2.0 5.0 10
sCLU
Ku70
C UT 10 Gy UT 10 Gy 96 4 24 48 72 96 96 4 24 48 72 96
sCLU 1.8 kb
0.7 kb 36B4
Fold Induction: 1 <1 <1 <1 <1 1.5 1 1 1 3 6 7
Figure 2.3. sCLU is induced in HCT116:p53-/- cells, but not in p53+/+
HCT116:parental cells. Asynchronous HCT116:parental and HCT116:p53-/- cells were exposed to 10 Gy and protein harvested at various times. In A, western blot analyses were performed as in Figure 2.2, and probed for sCLU, p53 and Ku70 as described in Experimental Procedures. In B, HCT116:p53-/- cells were treated with various doses of IR and protein was harvested 72 h later. Western blot analyses were performed as in Figure 2.2. In C, total RNA (10 µg) was analyzed using
77 northern blot techniques as described in Figure 2.1 and Experimental Procedures.
Shown are representative blots from experiments performed at least three times.
78 sCLU is not cell cycle regulated.
An alternative explanation for sCLU induction and subsequent repression by p53 could be that the CLU gene is cell cycle regulated, and that wild-type p53-expressing cells suppress sCLU expression by arresting cells in a particular phase of the cell cycle.
Recent reports suggested that sCLU may be expressed exclusively in quiescent normal cells (328). To address this issue in cancer cells, HCT116:parental, HCT116:p53-/- and
-/- HCT116:p21 cells were arrested in the G0/G1 phase of the cell cycle by dual serum- starvation and confluence-arrest conditions, released by replating and irradiated 10 h later as described in ‘Experimental Procedures’ (329). The cell cycle profiles of untreated and irradiated isogenic HCT116 cells were then monitored (Figure 2.5). Untreated
HCT116:parental cells subsequently entered S-phase 14-16 h after release from low
serum and confluence arrest, with concomitant decreases in G0/G1 cells.
As previously noted with this synchronization technique (330), one synchronous cell division was achieved, and mock-irradiated isogenic HCT116 cells returned to a log- phase cell cycle distribution after 58 h post-release. There were significant differences in synchronized mock-irradiated HCT116:parental, HCT116:p53-/- and HCT116:p21-/- cells, particularly in the time of entry into S-phase, with both p53- and p21-deficient HCT116 cells entering S-phase sooner than wild-type p53 HCT116:parental cells (compare the
cell cycle distributions in Figures 2.5A, C and E). Since p53 exerts its G1 cell cycle checkpoint responses through, in part, induction of p21, mock-irradiated and IR-treated
HCT116:p53-/- and HCT116:p21-/- cell cycle distributions were very similar (Figures
2.5C, E).
79 A HCT116:p21-/- UT 10 Gy Time (h): 96 4 24 48 72 96
sCLU
p53
Ku70 B UT 10 Gy Time (h): 4 96 4 24 48 72 96
sCLU 1.8 kb
36B4 0.7 kb
Fold Induction: 1 <1 <1 <1 <1 <1 <1
Figure 2.4. sCLU is not induced in HCT116:p21-/- cells. HCT116:p21-/- cells were exposed to 10 Gy and protein harvested at various times. In A, western blot analyses were performed as in Figure 2.2. Blots were probed for sCLU, p53 and
Ku70 by western blot analyses as described in Experimental Procedures. In B, total
RNA (20 µg) was analyzed using standard northern blot techniques as described in
Figure 2.1. Shown are representative blots from experiments performed at least three times.
80 As expected, p53+/+ HCT116:parental cells treated with 10 Gy at 10 h post release resulted in a significant delay in the progression of synchronized cells into S-phase (a
function of the IR-induced G1 cell cycle checkpoint response, Figures 2.5A, B) as described (330). For example, at 36 h IR-treated HCT116:parental cells demonstrated
>45% G2 cells compared to less than 18% in mock-irradiated cells (compare Figures
2.5A, B). In contrast, HCT116 cells with somatic deletions of p53 (Figure 2.5C, D) or p21 (Figures 2.5E, F), entered S-phase earlier, with accumulation of S-phase cell
populations occurring at 12-16 h, accompanied by concomitant decreases in G0/G1 cells.
At 18 h after release (8 h after 10 Gy IR exposure), only 14% and 28% of p53-/- and p21-/-
cells, respectively, remained in G1, while 80% and 66% of cells, respectively, proceeded
+/+ into S phase. As expected, IR-treated HCT116:p53 cells arrested in G1 and exhibited
-/- -/- delayed S or G2/M phase entry compared to IR-treated HCT116:p53 or HCT116:p21 cells. HCT116:p53-/- and HCT116:p21-/- cells responded similarly to IR treatment.
Although responses to 10 Gy are shown, near identical responses to 2-5 Gy were also observed in other studies (330).
Western blot analyses of non-irradiated synchronized HCT116:parental,
HCT116:p53-/- and HCT116:p21-/- cell populations indicated that the levels of sCLU did not change relative to basal levels throughout the cell cycle (Figure 2.5). Interestingly, sCLU was induced only in IR-exposed synchronized HCT116:p53-/- cells with similar induction kinetics (maximal accumulation observed between 24-72 h) as noted in IR- treated asynchronous log-phase HCT116:p53-/- cells (Figure 2.3). In contrast, only minimal sCLU induction responses were noted in synchronized IR-treated
HCT116:parental or HCT116:p21-/- cells, even though irradiated HCT116:p21-/- cells
81 exhibited nearly identical cell cycle distribution changes as IR-exposed HCT116:p53-/- cells. These data strongly suggested that: (a) sCLU induction was genetically programmed after IR stress resulting in a 48 – 72 h delay before sCLU accumulation was noted after IR exposure; and (b) sCLU was transcriptionally repressed by functional p53 independent of the cell cycle. Loss of functional p53 appears to relieve negative regulation on the IR-induction responses of CLU gene expression in a variety of cell types.
DISCUSSION
Our laboratory previously demonstrated that sCLU was an x-ray induced protein (i.e., xip8) (35). In this study, we further investigated the induction of sCLU by IR. We showed that sCLU was induced by doses of IR as low as 2 cGy (Figures 2.1A & B).
This low-dose IR induction is seen at both the transcript and protein levels, with promoter activation noted after 0.5 Gy in MCF-7 cells containing a stably integrated 1403 bp human CLU promoter directing expression of firefly luciferase. Induction of the CLU promoter was noted only after 0.5 Gy as analyzed by luciferase assays using a luminometer (Figures 2.1C & D), however, induction of this promoter after IR doses <50 cGy has been noted when analyzed by bioluminescent imaging (Klokov et al., unpublished data). We also showed that DNA damage appears to be sufficient, but not required for sCLU induction (Table 2.1).
We demonstrated that the basal level and IR induction of sCLU after IR exposure was repressed by p53. MCF-7 and RKO cells stably expressing the HPV E6 protein
(both exhibiting loss of functional p53) have high basal levels of sCLU compared to
82 parental cells that express functional p53 (Figure 2.2). Additionally, HCT116:parental and RKO:neo cells that express wild-type p53 minimally induce sCLU after IR, whereas
HCT116:p53-/- and RKO:E6 greatly induced sCLU at the protein and transcript level after
IR (Figure 2.3). Finally, the effect of p53 on IR-inducible sCLU expression was not dependent on the cell cycle, but was delayed in its induction, requiring at least 48 h post-
IR in all cells examined (Figure 2.5). Since the relationship between p53 status and sCLU expression and p53 repression of IR-induced sCLU expression was observed in cells of different origins, p53 repression of this gene appears to be a general phenotype and not unique to specific cell lines. A limited screen of cancer cell lines indicated an inverse regulatory relationship between p53 status and sCLU expression (Table 2.2). To directly explore the role of p53 in basal and IR-inducible levels of CLU gene expression, we used three model cell line systems from breast and colon cancer origins to investigate the role of p53 in the transcriptional regulation of sCLU. All three cell lines confirmed that p53 exerted negative regulation on sCLU expression.
The effect of IR exposure on sCLU expression in MCF-7 cells was different from that found in HCT116 parental cells, even though both cell lines express wild-type p53. HCT116:parental cells did not induce sCLU after IR exposure. In fact,
MCF-7 cells are the only wild-type p53-expressing cell line examined to date that strongly induced sCLU after IR. It may be that MCF-7 cells over-express the IR- activated transcription factors required for induction of CLU gene expression, while
HCT116 cells maintain lower levels, which are in turn efficiently suppressed by wild- type p53 even after IR exposures. It appears that these as yet unknown transcription
83 A B HCT116:parental 100 parental 100 IR UT 80 80 60 60 40 40 20 20 0 0 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Percent Cell Number Time (h) after 5 Gy MCF-7 0 10release 18 (h) 24 36 58 5 Gy: - + - - - + - + - + - + CLU
p53
Ku70
C HCT116:p53-/- D 100 -/- 100 IR UT p53 80 80 60 60 40 40 20 20 0 0 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Percent Cell Number Time (h) after 5 Gy MCF-7 0 release 10 18 (h) 24 36 58 5 Gy: - + - - - + - + - + - + CLU
p53
Ku70
HCT116:p21-/- E p21-/- F 100 IR 100 UT 80 80 60 60 40 40 20 20 0 0 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Percent Cell Number Time (h) after 5 Gy MCF-7 0 release 10 18 (h)24 36 58 5 Gy: - + - - - + - + - + - +
CLU
p53
Ku70
84 Figure 2.5. sCLU is not cell cycle regulated. HCT116:parental, p53-/- and p21-/- cells were synchronized by serum starvation and confluence-arrest. Synchronized cells were released by low density seeding in 10% FCS-DMEM medium and cells were allowed to proceed through the cell cycle for 10 h and then mock-irradiated
(Fig. 2.5A, C, E), or exposed to 10 Gy (Fig. 2.5B, D, F). Cells were allowed to
progress through G1 (), S () and G2/M () phases of the cell cycle. Protein was harvested for flow cytometric or western blot analyses at the indicated times as described previously in Experimental Procedures. Western blots were probed for
CLU, p53 or Ku70 expression as described in Experimental Procedures. Shown are data for HCT116:p53+/+ parental (A, B), HCT116:p53-/- (C, D) and HCT116:p21-/- (E,
F) cells. Western blots and cell cycle analyses are representative of experiments performed at least three times.
85 factors may be constitutively expressed in MCF-7 cells, since E6 expression greatly enhanced sCLU expression in MCF-7 cells without IR exposure, whereas loss of functional p53 in HCT116 cells did not cause an appreciable increase in basal levels of sCLU protein expression. The factors needed for sCLU induction have not been elucidated. Analyses of the transcription factors and DNA elements within the CLU promoter that regulate the IR inducibility of this gene are currently being performed in our laboratory.
The signaling pathway(s) that regulate sCLU induction and expression after IR exposure is(are) unknown. Our laboratory identified CLU as a Ku70 binding protein using yeast-two-hybrid analyses (71). Through our screen of cytotoxic agents, we noted that DNA damage was not required for sCLU induction. This was best demonstrated by the induction of sCLU after thapsigargin (TG) exposures, and at doses of TG (2 nM, 1 h) that are not lethal to exposed MCF-7 cells (Table 2.1). TG is an inhibitor of the SERCA pump in the ER. Treatment of MCF-7 cells with TG resulted in a transient release of intracellular calcium (331) and an induction of sCLU mRNA and protein, suggesting that calcium changes may be an upstream signaling event mediating sCLU induction. It is possible that calcium, as a signaling molecule, may be a triggering event common to all the agents in Table 1 that elicit sCLU induction responses. The exact signal transduction processes that result in CLU gene expression after DNA damaging agents compared to non-DNA damaging agents is being elucidated in our laboratory.
The mechanism of sCLU repression by p53 also remains to be elucidated. There are several proposed models of p53 transcriptional repression. In the first model, p53 binds to its putative DNA binding sequence and sterically inhibits the binding of
86 transcription factors required for induction. This model was proposed to account for repression of Bcl-2 (332), α-fetoprotein (333) and HBV (334) genes by p53. In the second model, p53 binds and sequesters transcription factors required for upregulation.
For example, p53 can directly bind several transcription factors including Sp-1 (335,
336), AP-1 (337), NF-Y (338, 339), Brn-3a (332) and C/EBPβ (340), that may be responsible for upregulated CLU promoter activity after IR. Additionally, it was shown that p53 can bind the TATA binding protein (TBP) in vitro and inhibit transcription by disrupting formation of the TFIID complex (341). Alternatively, Johnson et al. have proposed a novel putative DNA binding sequence for p53 that is strictly involved in transcriptional repression (342).
Collectively, our data strongly suggest that the CLU gene is transcriptionally repressed by p53, although the mechanism of this repression still remains to be elucidated. The cell models used in this study will allow us to further investigate the mechanism(s) of p53 repression of sCLU, as well as the signaling pathways required for sCLU induction after IR exposure. Understanding the cellular responses to ionizing radiation exposure, in normal and tumor tissue, is vital for improving the efficacy of radio-therapy in the clinic.
The data presented in this paper provide a first examination of how a cell may regulate the clusterin molecular switch, turning on the cytoprotective sCLU protein at low doses of IR (0.02 - 0.1 Gy), while at the same time allowing p53 responses after high doses of IR (≥ 1.0 Gy) to shut down this cytoprotective protein to allow for cell cycle checkpoint responses and for cell death in severely damaged cells. For example, we are exploring the possibility that functional p53 is responsible for mediating CLU alternative
87 splicing that produces nCLU protein expression (95). We previously demonstrated that nCLU, and not sCLU, could associate with Ku70 and cause apoptotic cell death responses. In this way, p53 would down-regulate the cytoprotective sCLU protein, while simultaneously stimulating the synthesis and possibly activation of nCLU. Expression, and nuclear translocation, of nCLU after >1 Gy of IR would then result in a cascade of events leading to cell death and apoptosis. Understanding the regulatory events affecting the relative levels of different forms of the CLU protein after IR should allow elucidation of ways to modulate death responses in tumor cells, while possibly sparing the survival of normal cells.
88 Chapter 3: Induction of Clusterin, a Pro-Survival Factor, Requires Delayed
Activation of IGF-1R/MAPK Signal Cascade after IR
Submitted to Molecular Cell, March 2004
Abstract
Radiation therapy is a common treatment for many tumor types, yet the responsiveness to this treatment is variable. We show that secretory clusterin (sCLU), an ionizing radiation
(IR) – induced gene provides cytoprotection after IR. Induction of sCLU is a delayed event taking 72 hrs after radiation to reach maximal levels. This delayed induction is dependent upon the up-regulation and activation of insulin-like growth factor-1 receptor
(IGF-1R) and the downstream Src-Raf-Mek-Erk signaling cascade. This pathway culminates in the activation of early growth response-1 (Egr-1) that is required for sCLU induction. Thus, our data provides a mechanism by which cancer cells may provide cytoprotection for neighboring cells through potential bystander effects, and the development of radio-resistance after clinically relevant doses of IR through the IGF-1R-
Src-Mek-Erk-Egr-1 cascade.
89 Introduction
Ionizing radiation (IR) is a common therapy for many types of cancers, and elucidation of refractory responses to clinically relevant doses of IR are under intense investigation.
Understanding these cellular processes, especially the contribution of signal transduction pathways that precede gene expression after IR will lead to interventions that bring about a more favorable clinical outcome. Although it was once thought that the only important cellular responses to IR originated from DNA damage, it is clear that IR creates many different lesions in macromolecular targets within the cell that set in motion cascades of responses in both irradiated, as well as in neighboring non-irradiated cells. For example, the epidermal growth factor receptor (EGFR) (343, 344) , insulin-like growth factor-1 receptor (IGF-1R) (345, 346) and the tumor necrosis factor-α (TNF-α) receptor (347) have been reported to be activated after IR resulting in intracellular signaling cascades that lead to gene expression that mediates resistance to IR-induced cell killing (348). The epidermal growth factor (EGF) family of tyrosine kinase receptors, including EGFR (also called Erb1), Erb2, Erb3 and Erb4, are thought to mediate cell survival after IR exposure through the activation of the MAPK cascade (344, 349). IGF-1R over-expression can also mediate radio-resistance through the activation of the Mek/Erk and PI-3 kinase pathways (348). Furthermore, the TNF-α receptor is thought to mediate radio-resistance by activation of NF-κB (350). IR also increases production of the ligands for these receptors (e.g., EGF, TGF-α, IGF-1, and TNF-α) resulting in activation of the receptor and induction of downstream signaling pathways (345, 349, 351, 352). Since these signaling cascades can enhance the survival of cancer cells, their stimulation represents pathways that limit the effectiveness of radio-therapy in the treatment of a variety of
90 human malignancies that over-express these receptors. Thus, elucidating mechanisms that may be targeted by inhibitors in combination with radiotherapy should improve therapeutic efficacy. Indeed, small molecule inhibitors and antibodies to the EGFR family members are already in clinical trials (353-357), but these therapies will only be effective if EGFR is the major component of protection and resistance.
Clusterin (CLU) is a highly glycosylated secretory protein thought to provide cytoprotection after various cell stresses due to its role as a molecular chaperone. sCLU has been shown to clear and mediate the turn-over of extracellular debris (75, 76, 78, 325,
358). CLU plays a direct role as a cellular stress sensor, since a variety of cytotoxic agents (topotecan, etoposide, colcemid, nocodazole, taxol and thapsigargin) induce CLU gene and protein expression (94) at doses far below their cytotoxic effects.
CLU has also been implicated in many normal biological processes including tissue remodeling (21, 359, 360), sperm maturation (361, 362), complement inhibition
(15, 305, 363, 364), lipid transport (9, 13, 365), and pathological states ranging from
Alzheimer disease (39, 309, 366, 367), atherosclerosis (37, 307, 308, 368, 369), prion diseases (370-372), rheumatoid arthritis (45, 373), glomerulonephritis (82, 374, 375) and many cancers (51, 53, 57, 59, 376-379). Over-expression of endogenous CLU correlates with higher tumor grade and poor prognosis in prostate and breast cancer (49, 51, 58,
377). Additionally, over-expression of exogenous CLU results in resistance to paclitaxel
(62, 310), doxorubicin (87), cisplatin (63) and radiation therapy (380), while antisense
CLU expression enhances the chemosensitivity of various cell lines (73, 74). Together these data suggest that sCLU provides a survival advantage for the cancer cells that over-
91 express the endogenous protein. Thus decreasing or eliminating sCLU levels during various cancer therapies may increase the efficacy of these treatments.
Our laboratory identified CLU as an IR-induced protein/transcript (91). Secretory clusterin (sCLU), the fully processed, glycosylated form of this protein is induced by IR doses as low as 2 cGy (35, 94). Interestingly, peak CLU promoter activity, transcript and protein levels occurred 48-72h after IR exposure (94). To date, the signaling cascades leading to CLU upregulation after IR remain undetermined. Several transcription factors have been implicated in sCLU induction after various stress responses, including c-fos
(86), c-myb (87) and the TGF-β pathway (84-86), although the signaling pathways involved in the induction after IR have not been elucidated.
In this report, we show that sCLU is involved in cell survival after IR, by siRNA.
Furthermore, the activation of the Src-Raf-Mek-Erk signaling cascade, by clinically relevant doses of IR, in MCF-7 breast cancer cells is required for sCLU induction.
MAPK signaling was detected in cells within hours after IR as previously reported (344,
348, 349). However, we discovered a dramatic re-activation of MAPK signaling 24-72 h after IR. This delayed activation of MAPK was required for the late induction of the
CLU promoter, and subsequent elevations in sCLU transcript and protein. In defining the temporal and dose-response relationships of activation of this delayed signal transduction pathway, we identified a role for the insulin-like growth factor-1 receptor (IGF-1R) in the induction of sCLU after IR. Since sCLU has been shown to be a cytoprotective protein, these results may explain the radio-resistant phenotype of tumor cells that over-express the IGF receptor (345, 348, 352). A better understanding of the signal transduction
92 pathways activated by IR that stimulate sCLU levels in cells will allow for improved efficacy of radiation therapy against many human malignancies.
Experimental Procedures
Plasmids
The Src CA, Src KD, dn Erk-1, dn Erk-2 and Egr-1 plasmids were a generous gift from
Dr. Lindsey Mayo at Case Western Reserve University. The dn Mek-1 plasmid was a kind gift from Dr. Jeff Holt at Vanderbilt University (381). The 4250 CLU promoter was constructed as follows. The following primers were used to amplify the 4676 bp fragment of human CLU promoter:
5’ TGG CTA GCC TCA GAC CTT TAC CCA AGA AGA TC 3’
5’ GCT CGA GCG TTG TGG GCA CTG GGA G 3’
Genomic DNA from MCF-7 cells was used as a template. PCR was performed using
Elongase enzyme mixture (Invitrogen) with 1.8-2 mM of MgCl2. PCR conditions were:
94˚C – 45 sec; 63˚C – 30 sec; 68˚C – 5 min; 35 cycles. The 4676 bp product was gel
purified, and a 4250 bp was inserted into the SmaI site of pA3luc plasmid (a gift from Dr.
Richard G. Pestell, Georgetown University, Washington, DC). The orientation of the insert was verified by restriction digest and partial sequencing (Leskov et al., unpublished data).
93 Cell Culture and Transfections
MCF-7 breast cancer cells were grown in RPMI 1640 with 5% FBS at 37˚C in a humidified incubator with 5% CO2-95% air atmosphere. Media was replenished every 24 h on experimental plates.
PP1 was obtained from BioMol (Plymouth Meeting, PA) and U0126 was obtained from Cell Signaling Technology (Beverly, MA). AG1478 and AG1024 were obtained from Calbiochem (La Jolla, CA). EGF and IGF were obtained from Sigma or
Calbiochem respectively. EGF/IGF experiments were performed as follows. Cells were plated at a confluency of 5 X 105 cells per 10 cm2 plate. Twenty-four hours later, normal growth media was removed and replaced with media containing no serum. The next day, cells were pretreated for 1 h with either AG1478, AG1024 or DMSO and then were exposed to 5 Gy ionizing radiation with 137Cs gamma rays at a dose rate of 0.87-0.92
Gy/min, using a Shepard Mark Irradiator or treated with IGF or EGF in media containing no serum. One hour after radiation or IGF/EGF treatment, cells were replated into media containing 1% serum and were maintained in 1% serum until harvest at 48 h. AG1478 and AG1024 were removed 24 h after IR or IGF/EGF treatment.
PP1 and U0126 experiments were performed as follows. Cells were plated at a confluency of 5 X 105 cells per 10 cm2 plate. The next day, cells were pretreated for 1 h with the either PP1, U0126 or DMSO then exposed to 5 Gy. Untreated cells were mock- irradiated as described (11). In Figures 5A and 5B, media containing 20 µM PP1 was removed from the cells 24 h after IR. In all other experiments a continuous exposure of
PP1 or U0126 was used, and new PP1 or U0126 in fresh media was added to the cells every 24 h until harvest at 72 h.
94 The 1403 cell line stably expressing 1403 bp of the CLU promoter linked to the luciferase gene was generated as previously described (94). MCF-7 cells were transiently transfected using Effectene (Qiagen; Valencia, CA), according to the manufacturers directions.
Luciferase Assays
Luciferase assays with the 1403 CLU promoter were performed with the Luciferase
Assay System (Promega; Madison, WI). Cells were seeded in 6-well plates at approximately 50% confluency. Cells were treated with inhibitor and/or 5 Gy and harvested at the indicated time point in 1X reporter lysis buffer (Promega; Madison, WI).
Luciferase assays performed on MCF-7 cells transiently transfected with the 4250 CLU promoter and Src CA, Src KD, dn Mek-1 or dn Erk-1/2 plasmids were performed using the Dual Luciferase System (Promega; Madison, WI). MCF-7 cells were transfected 24 h before treatment with inhibitor and/or 5 Gy and harvested in 1X reporter lysis buffer at the indicated times. All experiments were equalized for protein using Bradford Assays
(Bio-Rad Laboratories; Hercules, CA). Each dose/time point was completed at least in triplicate and a Student’s T-Test was performed to determine statistical significance.
Western Blot Analyses and Co-Immunoprecipitations
Whole cell extracts were extracted in RIPA buffer (0.1% SDS, 0.5% deoxycholate, 1%
NP-40, 150 mM NaCl, 50 mM Tris pH 8.0) and separated on a 12% gel by SDS-PAGE western blot analyses as previously described (35). Proteins were transferred to
Immobilon-P (Millipore; Bedford, PA) and probed with antibody. Antibodies to human
95 sCLU (B5), Ku70 (C-19), P-Raf-1 (Ser338), Raf-1 (C-12), P-Mek1/2 (Ser218/222), Mek 1 (C-
18), P-Erk (E-4), Erk 1 (K-23), P-Fak (Tyr925), Egr-1 (588) and Sp1 (PEP2) were obtained from Santa Cruz (Santa Cruz, CA). IGF-1 receptor α, P-EGF receptor (Tyr1068),
EGF receptor and c-Src (Tyr416) antibodies were obtained from Cell Signaling
Technology (Beverly, MA). α-tubulin and c-Src (Tyr416) antibodies were obtained from
Calbiochem (La Jolla, CA).
EGFR was immunoprecipitated from 250 µg total protein using 2 µg antibody at
4˚C overnight. Complexed lysates and antibodies were incubated with protein G/agarose beads at 4˚C for 1 h. Complexes were washed three times with RIPA buffer, resuspended in 25 µl of SDS loading buffer, boiled for 5 minutes and separated on a 12% SDS-PAGE gel.
Quantitation was performed using NIH Image. Relative levels were determined by dividing the experimental protein levels by the total protein levels.
siRNA and Clonogenic Survival Assays
The following siRNAs against sCLU mRNA (siRNA-sCLU) or to a scrambled sequence were synthesized by Dharmacon, Inc. (Lafayette, CO): siRNA-sCLU 5'– GCG UGC AAA GAC UCC AGA AdTdT–3'
3'–dTdTCGC ACG UUU CUG AGG UCU U–5' siRNA-scrambled 5’-GCG CGC UUU GUA GGA UUC GdTdT-3’ 3’-dTdTCGC GCG AAA CAU CCU AAG C-5’
5 ug of siRNA-CLU, or scrambled siRNA was transfected into MCF-7 cells (5x105 cells/60 mm dish) using Lipofectamine Plus (Invitrogen, Inc.; Carlsbad, CA) according to the manufacturer’s instructions. Mock-transfected cells were used as a control. 48 h after
96 transfection, cells were trypsinized, and plated onto 60 mm dishes (500 cells per dish) in triplicates. Ten days later, cells were fixed and stained using crystal violet. Colonies containing >50 normal looking cells were counted.
siRNA to Egr-1 was generated by Dharmacon, Inc. (Lafayette, CO) as a Custom
SMARTpool that contained four siRNA sequences against Egr-1. MCF-7 cells were transfected with 20 µM siRNA-Egr-1, 20 µM scrambled siRNA or mock transfected using Oligofectamine following the manufacturers protocol (Invitrogen, Inc; Carlsbad,
CA). Twenty-four hours after transfection, cells were exposed to 5 Gy or mock- irradiated. Cells were harvested in 1 X reporter lysis buffer for luciferase assays or RIPA buffer for western blot analyses.
DNA Pull-Down Assays
A biotinylated full length CLU promoter was amplified from the 1403 pA3luc plasmid using the following primers ordered form Integrated DNA Technologies (Coralville, IA):
5’ GAT CCA TTC CCG ATT CCT 3’
5’ /5Bio/ AGC CAA GCT TCC TGT GCC 3’.
Nuclear extracts were harvested from DMSO, PP1 or U0126 treated MCF-7 cells that were either treated with 5 Gy or mock-irradiated as previously described (382). Three µg of the biotinylated CLU promoter were incubated with 10 µl of strepavidin beads
(Oncogene Research Products; Boston, MA) for 1 h at room temperature. The complex
was washed in binding buffer (50 mM Tris-HCL pH 7.5, 5 mM MgCl2, 2.5 mM EDTA,
2.5 mM DTT, 250 mM NaCl, 0.25 µg/µl poly (dI-dC)· poly (dI-dC) and 20% glycerol) before the addition of 100 µg nuclear extract and binding buffer up to 20 µl and
97 incubated at room temperature for 20 minutes. One ml of binding buffer was added and the complexes were incubated at 4˚C overnight. 24 h later, complexes were washed twice in binding buffer, beads were resuspended in 20 µl of SDS-PAGE loading buffer and separated on a 12% gel.
Results
siRNA to sCLU in MCF-7 Cells Causes Radio-sensitization
It has been reported that induction of sCLU by various chemotherapeutic agents provides cytoprotection for the cell (73, 74, 87). To determine if sCLU provides a protective role after IR, siRNA specific to the leader peptide in sCLU was transiently transfected into MCF-7 cells. sCLU protein levels were decreased 2-fold in MCF-7 cells using siRNA to sCLU (Figure 3.1B).
Decreased sCLU protein levels resulted in a significant increase in the lethality of cells treated with increasing doses IR, as determined by clonogenic survival assays (Figure 3.1A). In contrast, mock-transfected or scrambled siRNA (scr-siRNA) did not alter the survival of irradiated MCF-7 cells (Figure 3.1A), or sCLU protein levels (Figure 3.1B). These results illustrate the cytoprotective role of sCLU in irradiated MCF-7 cells, and suggest that alterations that decrease sCLU induction could alleviate the survival advantage after IR that sCLU provides cells.
98 A
1
0.1 * * 0.01 Mock
Surviving Fraction Scr-siRNA * SCLU-siRNA 0.001 * 0 2 4 6
Dose (Gy) *p<0.01
B Mock- scrambled sCLU transfected siRNA siRNA
sCLU
α-tubulin
Relative Levels: 1 0.9 0.5
Figure 3.1. siRNA knock-down of sCLU in MCF-7 Cells Causes Radio-sensitization.
(A) siRNA to sCLU increases radio-sensitivity of MCF-7 cells. Cells were transfected as described in “Experimental Procedures” and allowed to grow 10 days before being stained with crystal violet and counted for clonogenic survival assay.
(B) siRNA to sCLU diminishes sCLU protein expression. siRNA-sCLU was transfected into MCF-7 cells. Protein was harvested 60 h later and separated by
SDS-PAGE. Relative levels were determined as compared to mock-transfected controls.
99 Activation of MAPK by IR
It is generally presumed that the growth factor induced signaling pathways are activated and then shutdown very quickly after a given stimulus. Since sCLU is expressed late after
IR, with induction occurring 48-72 h after exposure (94), it was not understood how early signaling events could result in the late activation of this gene. To examine the temporal activation of kinases that may correlate with sCLU induction, we used western blot analyses to examine the activation kinetics of Src, Raf, and Mek in MCF-7 cells after 5
Gy (Figure 3.2).
Phospho-specific antibodies that indicated activation of these proteins, were compared to changes in total protein levels. P-Src (Tyr416), P-Raf (Ser338), P-Mek-1/2
(Ser218/222) and P-Erk-1/2 (Tyr204) were detected after IR at 0.25 h, 0.5 h, 0.5-1.0 h, and
0.25-2 h, respectively. Thus, as previously reported after high doses of IR (344), the
Src/Raf/Mek cascade was induced immediately after IR exposure. Surprisingly, however, a dramatic re-activation of the Src/Raf/Mek/Erk1/2 pathway was noted at 24-72 h after IR that appeared to correlate with sCLU protein induction in MCF-7 cells. Taken together, the data in Figure 3.2 show that the MAPK cascade was reactivated in MCF-7 cells starting at 24 h after IR in a temporal sequence that correlated with sCLU protein induction after IR.
EGFR is Not an Upstream Activator of Clusterin
Prior work demonstrated that IR can activate the epidermal growth factor receptor
(EGFR), resulting in activation of the Raf-Mek-Erk MAPK cascade (344, 349, 383). In light of this work, we examined the role of EGFR in mediating CLU expression after IR,
100 using 1403 MCF-7 breast cancer cells, as previously described (94). Serum-starved cells were pretreated with AG1478, a selective inhibitor of EGFR kinase activity, for 1 h and cells were then mock-irradiated or exposed to 5 Gy. Increasing doses of AG1478 did not affect IR-induction of the CLU promoter monitored 48 h post-IR. AG1478 did not affect basal CLU promoter activity (Figure 3.3A).
To determine if epidermal growth factor (EGF) could directly induce sCLU expression, CLU promoter activation and sCLU protein levels were examined after various doses of EGF. EGF did not induce CLU promoter activity over control levels, and various doses of AG1478 did not significantly affect CLU promoter activity before or after IR (Figure 3.3B). These results were confirmed by western blot analyses (Figure
3.3C), where sCLU was not induced by treatment with 1 µM AG1478 (Lane 4), although
EGF (1 ng/ml) induced sCLU protein levels (Lane 5) and this induction was also not blocked by treatment with AG1478 (Lane 6). We do not fully understand the mechanism of EGF induction of sCLU, but this could be a direct effect of other EGFR family members that are not inhibited by AG1478. We are currently investigating this. Since
AG1478 was able to attenuate EGFR activation (Figure 3.3D) but did not alter IR induction of sCLU, we can conclude that the EGFR does not play a role in the induction of sCLU after IR.
Inhibition of Insulin-Like Growth Factor-1 Receptor Abrogates Clusterin Induction after IR
Given that inhibition of EGFR by AG1478 had no effect on sCLU expression while EGF treatment of MCF-7 cells resulted in elevated sCLU protein levels, we examined the
101 Time (h) Post-IR 0 0.25 0.5 1 2 4 8 24 48 72 (h): P-src
src
Relative Levels 1.0 7.4 11.6 5.1 4.2 12.9 12.8 15.4 14.1 14.5 (P-Src):
P-Raf
Raf
Relative Levels 1.0 1.0 1.3 0.9 0.9 0.6 1.2 0.6 1.4 1.1 (P-Raf):
P-Mek
Mek
Relative Levels 1.0 0.6 0.4 0.5 0.7 0.6 0.8 1.8 1.6 2.5 (P-Mek):
P-Erk 1/2
Erk 1
Relative Levels 1.0 2.7 1.7 1.9 1.5 1.3 1.6 3.3 3.9 2.0 (P-Erk1/2):
SCLU (60 kDa)
Ku70
*Relative Levels 1.0 0.8 0.8 0.8 0.8 0.9 1.0 1.5 1.8 2.0 (sCLU):
Figure 3.2. Activation of MAPK by IR.
The MAPK cascade is activated in MCF-7 cells after exposure to 5 Gy. MCF-7 cells were exposed to 5 Gy and harvested for western blot analyses at the indicated time points. Western blot analyses were performed as described in “Experimental
Procedures”. Relative levels determined as irradiated samples compared to unirradiated control samples.
*Relative sCLU levels were calculated with respect to Ku70 levels.
102 A B 600 600 UT DMSO 500 5 Gy 500 AG1478 400 400
300 300 Promoter Activity Promoter Activity 200 200 CLU at 48h Post-IR CLU at 48h Post-IR 100 100
0 1
Relative 0
0 0.5 1.0 5.0 0 0.5 1.0 5.0 Relative 1 0 0.5 1.0 5.0 0 0.5 1.0 5.0 AG1478 Dose (µM) EGF Dose (ng/ml)
C IR (5 Gy) - + + - - -
AG1478 (1.0 µM) - - + + - +
EGF (1.0 ng/ml) - - - - + + sCLU Ku70 Fold Induction: 1 2 2 1 2 2
D IP: EGFR IR (5 Gy) UT IR & AG1478 AG1478 EGF EGF & AG1478 WB: P-EGFR WB: EGFR Fold Induction: 1.0 1.1 0.6 1.3 2.0 1.3
103 Figure 3.3. EGFR is Not an Upstream Activator of Clusterin.
CLU promoter activity and protein expression were not affected by treatment with the EGFR inhibitor, AG1478. MCF-7 cells or 1403 cells were placed in 0% serum,
24 h before irradiation or addition of EGF. One hour prior to irradiation or addition of growth factor, cells were treated with DMSO or the indicated concentration of AG1478. One hour after irradiation or addition of growth factor, media was removed and fresh media containing 1% serum was added to the cells.
Cells were maintained in 1% serum until harvest. One day after irradiation or addition of growth factor, AG1478 was removed. All samples were harvested 48 h after irradiation or addition of growth factor. (A) Increasing doses of AG1478 are not able to abrogate CLU promoter activity. (B) Increasing amounts of EGF, with and without AG1478, have no effect on CLU promoter activity. (C) AG1478 is not able to abrogate sCLU protein induction 48 h after exposure to 5 Gy. (D) EGFR co- immunoprecipitation shows that EGFR is phosphorylated on Tyr1068 after exposure
to 5 Gy or addition of 1.0 ng/ml EGF, and that this phosphorylation can be blocked
by pretreatment with 1µM AG1478.
104 possible involvement of insulin-like growth factor receptor 1 (IGF-1R) in IR-mediated
CLU induction. The IGF-1R is a potent activator of the Raf-Mek-Erk MAPK cascade
(384-386), however, involvement of IGF-1R after IR has not been explored in detail.
Serum-starved 1403 cells were pretreated with increasing doses of AG1024, an IGF-1R kinase inhibitor, and either mock-irradiated or exposed to 5 Gy (Figure 3.4A). Serum- starvation of MCF-7 cells increased the basal levels of sCLU promoter activity.
Treatment of cells with AG1024 resulted in significantly reduced CLU promoter activity compared to DMSO controls (Figure 3.4A). Interestingly, induction of the 1403 CLU promoter was not observed with increasing doses of IGF. Furthermore, when cells were pretreated with 2.5 µM AG1024, the observed elevated basal level of CLU promoter activity was abrogated (Figure 3.4B), suggesting a direct role for IGF-1R signaling in
CLU gene expression. These results were then confirmed at the protein level by western blot analyses (Figure 3.4C). As previously noted, sCLU was induced over basal levels
(Lane 1) by 5 Gy (Lane 2), and this induction was blocked by pre-treatment with 2.5 µM
AG1024 (Lane 3, Figure 3.4C). Treatment of MCF-7 cells with AG1024 alone had little affect on basal sCLU protein levels (Lane 4). IGF (10 nM) had no effect on sCLU protein expression in MCF-7 cells (Lane 5), but basal levels were reduced by pre-treatment with
AG1024 (Lane 6). Interestingly, we noted that IGF-1R was slightly induced in MCF-7
105 A B 500 800 450 UT 700 DMSO 400 5 Gy 350 600 AG1024 300 500 250 400 200 *
Promoter Activity * 300 150 * Promoter Activity 100 200 CLU at 48h Post-IR CLU at 48h Post-IR * * * 50 100 * 0 1 0 0 1.0 2.5 5.0 0 1.0 2.5 5.0 1 0 5 10 20 0 5 10 20 Relative AG1024 Dose (µM) Relative *p<0.04 IGF Dose (nM) *p<0.05
C IR (5 Gy ) - + + - - -
AG1024 (1.0 µM) - - + + - +
IGF (10 ng/ml) - - - - + +
sCLU
Ku70
Relative Induction: 1.0 1.6 1.1 1.1 1.0 0.8
D IR (5 Gy ) - + + - - -
AG1024 (1.0 µM) - - + + - +
IGF (10 ng/ml) - - - - + +
IGFR
Ku70
Relative Induction: 1.0 1.2 0.6 0.9 0.8 1.0
0.095
0.090 0.085 * 0.080 * *
0.075 IGF (ng/ml) 0.070 whole serum 0.065 serum starved
0.060 0 24 48 72 Time (h) Post-IR (5 Gy) *p<0.01
106 Figure 3.4. Inhibition of Insulin-Like Growth Factor-1 Receptor Abrogates
Clusterin Induction after IR. CLU promoter activation and protein expression were abrogated by treatment with the IGF-1R inhibitor, AG1024. MCF-7 cells or 1403 cells were placed in 0% serum, 24 h before irradiation or addition of IGF. One hour prior to irradiation or addition of growth factor, cells were treated with
DMSO or the indicated concentration of AG1024. One hour after irradiation or addition of growth factor, media was removed and fresh media containing 1% serum was added to the cells. Cells were maintained in 1% serum until harvest.
One day after irradiation or addition of growth factor, AG1024 was removed. All samples were harvested 48 h after irradiation or addition of growth factor. (A)
Basal and inducible CLU promoter activation is abrogated by pretreatment with increasing amounts of AG1024. (B) AG1024 decreases CLU promoter activity in serum-starved MCF-7 cells treated with increasing amounts of IGF. (C) Western blot analyses show that AG1024 is capable of abrogating sCLU protein induction after exposure to 5 Gy. (D) IGF-1R protein levels are upregulated in MCF-7 cells stimulated with either 5 Gy or 10 ng/ml IGF, and this up-regulation can be blocked by pretreatment with 2.5 µM AG1024. (E) An ELISA to human IGF-1 demonstrates that IGF-1 production and secretion is stimulated approximately 20% in MCF-7 cells 24-72 h after exposure to 5 Gy. *p value signifies statistical difference between irradiated samples and mock-irradiated samples.
107 cells after 5 Gy (Figure 3.4D). Treatment of cells with AG1024 and 5 Gy dramatically decreased IGF-1R levels (Lane 3). Addition of AG1024 also lowered the basal levels of
IGF-1R in unirradiated, serum starved cells, suggesting a possible autocrine feedback loop induced by IR, where irradiated cells up-regulate IGF-1R.
Since the receptor for IGF was elevated after IR, we investigated whether IGF-1 production was also elevated after exposure of cells to 5 Gy. Media collected from serum-starved MCF-7 cells at 0, 24, 48 and 72 h post-IR showed increased production of approximately 20% in irradiated MCF-7 cells at 24 h after IR, with sustained increases noted by 72 h post-IR (Figure 3.4E). These data suggest that MCF-7 cells have high basal levels of IGF-1 as reported (387), and that after exposure to 5 Gy, both he receptor and the ligand increase at later times (24-72 h) forming a putative autocrine feedback loop.
c-Src is an Upstream Activator of sCLU Induction After IR
It is known that c-Src, but not other Src family members, can activate and be activated by
IGF-1R (388-391). To determine whether c-Src activation was required for sCLU induction after IR, we used PP1, a selective inhibitor of Src family kinases. MCF-7 1403 cells were pretreated for 1 h with 20 µM PP1 and exposed to increasing doses of IR. PP1 was removed 24 h post-exposure, and luciferase activity was assessed at 72 h. PP1 abrogated induction of the sCLU promoter at low doses of IR, inhibiting sCLU induction by 50% at higher doses of IR (Figure 3.5A). As previously noted (35, 94), sCLU was modestly induced by 10 cGy in DMSO-treated cells, and this induction was blocked by
108 20 µM PP1 treatment (Figure 3.5B). PP1 inhibited IR-induced sCLU expression, with only minor inhibition noted after 2.5 Gy (Figure 3.5B), potentially due to the metabolism of PP1 by 72 h.
To address the potential loss of PP1 inhibition after a 24 h treatment period, we determined the lowest dose of PP1, given as a continuous 72 h treatment that could inhibit CLU promoter activity after 5 Gy. PP1 (5 µM) significantly decreased the basal activity of the CLU promoter and effectively blocked IR-activated CLU promoter activity
(Figure 3.5C). sCLU protein levels were also concomitantly decreased (Figure 3.5D). To confirm that the PP1 inhibitor was functional, we examined a known substrate of c-Src,
Fak. Fak is phosphorylated at Tyr925 by c-Src (391). Phosphorylation of Fak decreased with increasing doses of PP1 (Figure 3.5E). Thus, PP1 effectively blocked c-Src activity at doses (1 µM and 5 µM) that corresponded to doses used to block sCLU induction.
To confirm a role for c-Src as an upstream regulator of sCLU induction after IR, we transiently over-expressed increasing amounts of a constitutively active Src plasmid
(Y529F) or a kinase dead Src plasmid (K297R) in 1403 cells. Transfected 1403 cells were mock-irradiated or treated with 5 Gy and harvested at 72 h for CLU promoter activity. Titration of increasing amounts of constitutively active Src increased basal and
IR-inducible CLU promoter activity (lanes 1-6, Figure 3.5F). In contrast, titration of increasing amounts of kinase dead Src (Src KD) repressed IR-induced CLU promoter activity. Collectively, these data using PP1, a selective c-Src inhibitor, and expression of constitutively active or kinase dead Src, demonstrate the novel observation that c-Src is required for sCLU induction after IR.
109 A
1000 900 B 800 DMSO PP1 (20 M) 700 µ 600 IR (Gy): 0 0.1 0.25 0.5 1.0 2.5 0 0.1 0.25 0.5 1.0 2.5 500 * 400 sCLU Promoter Activity 300 200 Ku70 CLU at 72h Post-IR 100 0 IR (Gy): 0 0.1 0.25 0.5 1.0 2.5 5.0 1 0 0.1 0.25 0.5 1.0 2.5 5.0 PP1 (20 M) Relative DMSO µ *p<0.03
C 2.E+05 D 2.E+05 UT 2.E+05 1.E+05 5 Gy UT IR (5 Gy) 1.E+05 * 1.E+05 PP1 (µM): 0 1 5 10 0 1 5 10 8.E+04 * * 10 Promoter Activity * 6.E+04 sCLU
4.E+04 CLU at 72h Post-IR * * * 2.E+04 Ku70 0.E+00 1 0 1 5 10 15 0 1 5 10 15
Relative PP1 Dose (µM) *p<0.05
F E 800 700 * UT 600 * 5 Gy UT IR (5 Gy) 500
400 0 0 1 5 10 * * PP1 (µM): 300 * 200 Promoter Activity
P-Fak 100
CLU 0 at 72h Post-IR 1 1 2 3 4 5 6 7 8 9 10 11 Ku70 12 DNA (µg): Src CA Src KD Relative *p<0.05
110 Figure 5. c-Src is an Upstream Activator of sCLU Induction After IR
A 24 h exposure of MCF-7 cells to 20 µM PP1 can abrogate CLU promoter activation and sCLU protein expression after 5 Gy. (A) 20 µM PP1 can inhibit CLU promoter activation after increasing doses of IR. 1403 cells were pretreated with 20
µM PP1 one hour before irradiation. Media was removed, cells were washed with
PBS and fresh media without inhibitor was added 24 h after IR. (B) 20 µM PP1 can inhibit sCLU protein induction by IR doses as low as 0.1 Gy. MCF cells were treated as described above. (C) Basal and IR-inducible activity levels of the CLU promoter are inhibited by continuous treatment (over 72 h) of PP1. Increasing doses of PP1 were added to 1403 cells one hour before irradiation. Media with PP1 was replenished every 24 h. (D) sCLU protein induction by IR is abrogated in cells treated continuously with PP1. Increasing doses of PP1 were added to MCF-7 cells one hour before irradiation. Media with PP1 was replenished every 24 h.
(E) PP1 is able to inhibit phosphorylation of the Src downstream substrate, Fak, at doses that abrogate sCLU protein induction after 5 Gy. MCF-7 cells were pretreated for 1 h with increasing doses of PP1 and harvested for western 72 h after
IR. (F) MCF-7 cells transiently transfected with a plasmid expressing a constitutively active Src protein (Src CA) (Lanes 1-6) show an increase in basal and inducible promoter activity and cells transiently transfected with a kinase dead Src protein (Src KD) (Lanes 7-12) show inhibition of IR-inducible CLU promoter activity. Lanes 1, 4, 7 and 10 were transfected with vector only DNA. Lanes 2, 5, 8 and 11 were transfected with 0.2 µg of the appropriate DNA and lanes 3, 6, 9 and 12 were transfected with 0.3 µg of the appropriate DNA. All transfections contained a
111 total of 3 µg DNA. Cells were transfected 24 h prior to irradiation and harvested 72 h after IR.
Activation of the MAPK cascade is required for sCLU Induction After IR
Src phosphorylates the downstream signaling kinase, Raf, which in turn activates Mek-1 through phosphorylation (392). To examine the requirement of Mek-1 for sCLU activation, we used U0126, a selective Mek-1 kinase inhibitor, after IR. MCF-7 1403 cells were pretreated for 1h with increasing concentrations of U0126 and then exposed to
5 Gy. IR induction of the sCLU promoter was completely abrogated by 1µM U0126
(Figure 3.6A). Consistent with loss of gene induction, 1 µM U0126 completely abolished IR-induced sCLU protein in MCF-7 cells (Figure 3.6B).
The Erk 1/2 kinases are downstream substrates for Mek-1 (349). To demonstrate the involvement of Erks in CLU gene induction, we over-expressed dominant-negative
Erk-1 (dn Erk-1) or dominant-negative Erk-2 (dn Erk-2) in 1403 cells. After transfection, we mock-irradiated or exposed cells to 5 Gy and luciferase activity was examined 72 h later (Figure 3.6C). Both dn Erk-1 and dn Erk-2 completely suppressed CLU promoter activity after 5 Gy.
Log-phase MCF-7 cells were co-transfected with a 4250 bp CLU promoter- luciferase reporter and Src CA, Src KD, dominant-negative Mek-1 (dn Mek-1 K97A) or dominant-negative Erk-1 (dn Erk-1) (Figure 3.6D). In response to 5 Gy, the 4250 CLU promoter was induced approximately 4-fold in MCF-7 cells. Over-expression of constitutively active Src resulted in the elevation of the CLU promoter basal level 4-fold
112 A 1.E+04 B 1.E+04 UT * UT IR UT IR (5 Gy) 1.E+04 5 Gy 8.E+03 U0126 (µM): 0 0 5 10 1 5 10
Promoter Activity 20 6.E+03 sCLU
CLU 4.E+03 at 72h Post-IR Ku70 2.E+03
0.E+00 1 0 1 5 0 1 5 Relative U0126 Dose (µM) *p<0.05
C
250 UT 200 5 Gy E 150 Src Src VO CA KD Promoter Activity 100 * * * IR (5 Gy): - + - + - + CLU at 72h Post-IR WB: Src 50 WB: Ku70
Relative 0 dn 1 dn dn VO Erk-1 Erk-2 Erk-1/2 dn VO *p<0.02 Mek-1 IR (5 Gy): - + - + D WB: Mek-1 4.E+04 * WB: Ku70 4.E+04 UT 3.E+04 5 Gy 3.E+04 * dn VO Erk-1 Promoter Activity 2.E+04 2.E+04 IR (5 Gy): - + - + CLU at 72h Post-IR 1.E+04 WB: HA 5.E+03 * * * NB 0.E+00 1 Relative Src Src dn dn VO CA KD Mek-1 Erk-1 *p<0.05
113 Figure 3.6. Activation of the MAPK cascade is required for sCLU Induction After
IR. The Mek-Erk-Egr-1 pathway is required for sCLU induction after 5 Gy.
(A) The Mek-1 inhibitor, U0126, decreases IR-inducible CLU promoter activity to basal levels. 1403 cells were pretreated with increasing amounts of U)126 1 h prior to irradiation. Media with inhibitor was replenished every 24 h until cells were harvested 72 h after IR. (B) U0126 abrogates sCLU protein expression in
MCF-7 cells exposed to 5 Gy. MCF-7 cells were pretreated with increasing amounts of U)126 1 h prior to irradiation. Media with inhibitor was replenished every 24 h until cells were harvested 72 h after IR. (C) Transient transfection of dn Erk-1 or dn
Erk-2 into MCF-7 cells is able to diminish CLU promoter activity to basal levels after 5 Gy. (D) MCF-7 cells were transiently co-transfected with 4250 bp of the
CLU promoter in the pA3luc vector with either vector only (VO), constitutively active Src (Src CA), kinase dead Src (Src KD), dn Mek-1 or dn Erk-1. (E) Western blots confirming over-expression of Src, Mek-1 or Erk-1 in MCF-7 cells.
114 compared to control. Upon treatment with IR, a further increase in CLU promoter activity was observed in Src CA transfected cells. Over-expression of kinase dead Src, dominant-negative Mek- 1 and dominant-negative Erk-1 abrogated IR-induction of this promoter. Western blot analyses confirmed over-expression of the corresponding proteins from the plasmids used above (Figure 3.6E). These observations demonstrate that the
Src/Mek/Erk pathway was required for CLU promoter induction and sCLU protein expression in response to IR. Collectively, these data demonstrate the involvement of the
MAPK signaling cascade in sCLU induction in MCF-7 cells after IR.
The Egr-1 Transcription Factor can Bind to the CLU Promoter and is Required for
Promoter Activation After IR
There are several transcription factors that are known to be activated by the IGF-
1R/Src,Raf/Mek/Erk signaling cascade. Examination of the CLU promoter identified three potential Egr-1 binding sites (at –450 bp, -918 bp and –932 bp relative from ATG start site). Egr-1 is a known downstream target of the MAPK cascade (383, 393-396), and previously shown to be activated by high doses of IR (208, 283, 284, 286). Over- expression of Egr-1 increased CLU promoter activity~2-fold over basal levels, and an ~3- fold increase was noted in MCF-7 cells after exposure to 5 Gy (Figure 3.7A). Western blot analyses confirmed the over-expression of the Egr-1 protein in these cells (Figure
3.7B). This suggested to us that the Egr-1 protein is an important transcription factor that mediates induction of CLU gene expression after IR.
DNA pull-down assays were performed to determine if Egr-1 could bind to the
CLU promoter and if IR exposure of MCF-7 cells enhanced Egr-1 binding. The 1403
115 region of the promoter was amplified using a biotin-labeled primer complementary to the
3’ end of the CLU promoter. The biotinylated promoter fragment was bound to strepavidin beads, incubated with 100 ug of nuclear extract from MCF-7 cells treated with DMSO, 5 µM PP1 or 5 µM U0126 over 72 h (Figure 3.7C). After incubation, complexed DNA and proteins were prepared for western blot analyses. Egr-1 DNA binding activity was minimal in control mock-irradiated cells. After IR, however, an increase of Egr-1 binding was noted beginning at 4 h, with a robust increase in binding observed at 24 h, with sustained levels observed through 72 h post-irradiation. In contrast, IR-treated MCF-7 cells exposed to either 5 µM PP1 or 5 µM U0126 showed no increase in Egr-1 DNA binding to the CLU promoter DNA. Nuclear extracts (10% of input) were separated by SDS-PAGE and probed for PCNA as a loading control.
Since we were able to show that the Src/Mek/Erk pathway was involved in the induction of sCLU, we next determined if IGF-1R or EGFR inhibition could block Egr-1 binding to the CLU promoter. DNA pull-down assays were used to analyze Egr-1 binding to the CLU promoter in MCF-7 cells that were treated with DMSO, 1 µM
AG1024 (IGF-1R inhibitor) or 1 µM AG1478 (EGFR inhibitor) (Figure 3.7D). Exposure of IR-treated cells with AG1478 did not affect Egr-1 DNA binding activity to the CLU promoter. Consistent with our observations of IGF-1R mediating sCLU induction after
IR, Egr-1 DNA binding activity was blocked with AG1024. Interestingly, the basal Egr-
1 DNA binding to this promoter was increased in cells pretreated with either inhibitor compared to DMSO treated cells, which may be a result of cross-talk between the receptors. Competition assays using a non-biotin labeled CLU promoter (0.1 and 5 µg) showed that Egr-1 DNA binding activity was inhibited with increased concentrations of
116 unlabeled promoter, demonstrating specificity of Egr-1 DNA binding to the CLU promoter (Figure 3.7E).
To demonstrate the requirement of Egr-1 for sCLU induction, we used Egr-1 specific siRNA (Figures 3.7F & G). Consistent with previous results, IR exposure of mock-transfected 1403 cells or 1403 cells transfected with a scrambled siRNA (scr- siRNA) resulted in a 5-6-fold induction of the 1403 CLU promoter (Figure 3.7F). When control 1403 cells were transfected with 20 nM Egr-1 siRNA, the CLU promoter was not activated after 5 Gy. Efficacy of siRNA Egr-1 was confirmed by dramatic decreases in
Egr-1 protein levels (Figure 3.7G). Thus, decreased Egr-1 protein levels abrogated induction of CLU promoter activity at 72 h observed in 1403 cells after IR.
Discussion
Our studies revealed that sCLU plays a role in cell survival after exposure to IR
(Fig. 3.1). To further understand cellular responses to IR, we investigated the signaling pathways required for the IR-induction of sCLU. In this study, we report that IGF-1R activation after IR was required for sCLU induction. We demonstrated that IR-induced sCLU was dependent on a novel reactivation of the Src/Raf/Mek/Erk signaling cascade
24-72 h after IR exposure, and that this signal culminated in the activation of the Egr-1 transcription factor (model, Fig. 3.7H).
EGFR is over-expressed or constitutively activated in many types of tumors including colorectal, breast, pancreatic and ovarian cancers (397) and is known to be a mediator of radio-resistance in several tumor types including glioblastoma multiforme and breast cancer cells through the activation of Erk-1/2 (398, 399). As a result, many
117 A
7.E+04 * B 6.E+04 UT 5 Gy 5.E+04 VO Egr-1 * 4.E+04 IR (5 Gy): - + - + WB: Egr-1 3.E+04 Promoter Activity NB 2.E+04 CLU at 72h Post-IR
1.E+04
0.E+00 1
Relative VO Egr-1 *p<0.05
C DMSO PP1 (5 µM) U0126 (5 µM) Time Post-IR (h): 0 1 4 24 48 72 0 1 4 24 48 72 0 1 4 24 48 72
Egr-1
PCNA
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Fold 1818 18 Induction 1 1 2 6 3 7 1 2 1 1 1 1 1 2 1 1 1 0.4 :
1 µM 1 µM DMSO AG1024 AG1478 D UT IR UT IR UT IR
Egr-1
PCNA
1 2 3 4 5 6 Fold Induction: 1 2 1 0.5 1 1
E UT IR (5 Gy)
Egr-1
Non-biotinylated 1403 promoter (µg):
118 F 700 UT G 600 5 Gy Mock- Scrambled Egr-1 transfecte siRNA siRNA 500 d IR (5 - + - + - + Gy): 400 Egr-1
300 Ku70 Promoter Activity * Fold 200 Induction 1.0 1.8 1.1 1.9 1.2 0.9 CLU at 72h Post-IR : 100
0 1
Relative Scrambled Egr-1 Mock- *p<0.01 siRNA siRNA transfected (20 nM) (20 nM)
H
IR IGF-1R
IRS-1 SOS Src Grb2 Ras Raf
Mek-1
Erk-2 Erk-1
Erk-2 Erk-1
Egr-1 CLU promoter
119 Figure 3.7. The Egr-1 Transcription Factor can Bind to the CLU Promoter and is
Required for Promoter Induction After IR . Egr-1 binds to the CLU promoter and siRNA to Egr-1 abolishes CLU promoter induction by 5 Gy. (A) MCF-7 cells were transiently co-transfected with the 4250 CLU promoter and vector only (VO) or
Egr-1, 24 h prior to irradiation. Control cells were mock-irradiated (untreated,
UT). Luciferase assays were used to monitor CLU promoter activity 72 h after IR.
(B) Western blot confirming Egr-1 over-expression in MCF-7 cells. (C) DNA pull- down assay demonstrating the ability of Egr-1 to bind to the 1403 CLU promoter
(lanes 1-6) and the abrogation of this binding when cells are pretreated with 5 µM
PP1 (lanes7-12) or 5 µM U0126 (lanes 13-18) before 5 Gy. (D) The IGF-1R inhibitor, AG1024 (1 µM) diminished Egr-1 binding to the 1403 CLU promoter after exposure to 5 Gy as compared to DMSO treated cells. The EGFR inhibitor,
AG1478 (1µM) had no effect on Egr-1 binding to the 1403 CLU promoter after exposure to 5 Gy. (E) Egr-1 binding to the biotin-labeled 1403 CLU promoter can be competed away by increasing amounts (0.0, 0.1 or 5.0 µg) of non-biotin labeled
1403 CLU promoter.
(F) siRNA to Egr-1 lowers 1403 CLU promoter basal activity and IR-inducible activity as compared to mock-transfected cells or cells transfected with a scrambled siRNA. (G) Western blot analyses showing diminished Egr-1 protein levels in MCF-
7 cells after transfection with siRNA to Egr-1. Transfection with a scrambled siRNA had no effect on Egr-1 levels in MCF-7 cells. (H) Model depicting IR activation of
120 IGF-1R and the Src/Raf/Mek/Erk cascade culminating in the activation of Egr-1 and CLU promoter induction.
therapies have been developed that specifically target EGFR including monoclonal antibody therapies and small molecule inhibitors that specifically target the kinase domain (355). Interestingly, the selective EGFR inhibitor, AG1478, did not block CLU promoter induction or regulate sCLU protein levels after 5 Gy (Fig. 3.3), nor did it affect
EGF-stimulated sCLU protein expression, possibly due to the involvement of other
EGFR family members.
IGF-1R is another membrane receptor shown to be upregulated after IR. IGF-1R activation results in mitogenic growth and cell survival (384), and Gooch et al., demonstrated that treatment of cells with IGF-1 could prevent doxorubicin and taxol induced apoptosis (400). Using AG1024, a selective inhibitor of IGF-1R, we were able to block sCLU induction after IR (Fig. 3.4), demonstrating the requirement for IGF-1R activation for IR-induced sCLU expression. In a recent report, it was shown that
AG1024 treatment of MCF-7 cells enhanced cell death after IR exposure (401). Our data strongly suggests that activation of IGF-1R may mediate cell survival effects through the downstream induction of sCLU.
MCF-7 cells produce and secrete IGF-1 under serum-free conditions (387). In addition, IGF-1R is often over-expressed in breast cancer (402). It was shown previously that peripheral lymph node stromal cells produce and secrete EGF and IGF-1, which can increase the growth of breast cancer cells (403). It is possible that EGF and IGF secretion by lymph nodes can induce the tumorigenesis of neighboring breast tissue,
121 especially cells that have upregulated expression of EGFR or IGF-1R, through a paracrine mechanism. It is interesting to note that both EGF and IGF-1 were able to induce sCLU expression (Fig. 3.3C). Additionally, serum starvation increased the basal activity of the CLU promoter compared to cells grown in whole serum, suggesting a possible autocrine feedback loop induced by IR, where irradiated cells not only up- regulate IGF-1R, but also presumably increase production of the ligand, IGF-1.
Consistent with a previous report (384), we demonstrate induction of IGF-1R after IR
(Fig. 3.4D), as well as a 20% increase in secretion of IGF-1, 24-72 h post-IR (Fig. 3.4E).
Importantly, the induction of IGF-1R and its ligand provide a plausible explanation for the late induction of CLU after IR, as well as the increase in basal promoter activity after serum-starvation.
We show, for the first time, that the Src-Raf-Mek-Erk-1/2 pathway is required for
IR-induced sCLU activation. This cascade culminates in the activation of the Egr-1 transcription factor. In addition, we demonstrate a novel re-activation of the MAPK cascade after IR that correlates with the temporal activation of sCLU. The physiological relevance of this biphasic activation of MAPK is unknown. EGFR may be required for the initial induction of MAPK after IR, and inhibitors to EGFR are known to potentiate the cytotoxicity of radiation therapy (404). In addition, Lu et al., demonstrated that increased IGF-1R production in MCF-7 cells caused increased resistance to Herceptin (a monoclonal antibody to the Her2/neu receptor) induced cell death (405), suggesting a role for IGF-1R signaling in the development of resistance to this type of therapy. Our data suggests that tumor cells that survive the initial phase of treatment may develop resistance to EGFR inhibitors, as a result of the late induction of MAPK signaling
122 through IGF-R1 upregulation, and potentially the upregulation of sCLU. This suggests that the current antibody and small molecule therapies used to treat EGFR positive tumors may be optimized by the addition of inhibitors to the IGF-1R pathway.
sCLU has been shown to provide cytoprotection against doxorubicin, taxol and cisplatin in the treatment of cancer cells (63, 73, 74, 87). In this report, we show that sCLU provides cytoprotection in MCF-7 cells after IR exposure (Fig. 3.1). sCLU expression has been found to be elevated in many types of tumors, including prostate, colorectal and breast cancer (49, 51, 58). In a recent paper by Chen et al., it was shown that CLU message and protein were elevated in intestinal tumors derived from mice containing the Apc min (multiple intestinal neoplasia) mutation (59). IGF-1R and IGF-1 production are also elevated in many tumor types. Gleave et al., in a recent review, suggest using CLU and insulin-like growth factor binding proteins (IGFBPs) as targets for antisense therapy against prostate cancer (73), although they did not mention a possible connection between IGF-1R signaling and the induction of sCLU. Our data strongly suggests this connection.
It is intriguing to speculate a possible role for sCLU in bystander effects, after radiation or chemotherapeutic therapies. An increased production and secretion of sCLU by tumor cells into the lymph or vasculature system may provide a survival effect for neighboring or metastatic cancer cells. Additionally, secretion of IGF-1 by normal or tumor cells may provide a means for sCLU upregulation.
An increased understanding of the signaling pathways activated after IR will allow us to better determine which tumors will respond well to this type of therapy and which tumors will be refractive to radio-therapy. This is especially true for the delayed
123 cellular responses after IR that could have effects on carcinogenesis and cancer therapy.
Since sCLU provides resistance to IR, screening tumors for sCLU over-expression may provide a way of determining the efficacy of radio-therapy. Additionally, inhibitors to proteins in the signaling pathway leading to sCLU induction (i.e., IGF-1R/c-
Src/Raf/Mek/Erk pathway inhibitors) may be useful in treating human malignancies on their own and in combination with radiation.
124 Chapter 4: Conclusions and Future Directions
Radiotherapy is used to treat many types of cancers, but many tumors are refractory to this type of therapy or develop radio-resistance over time. A better understanding of the cellular responses to IR will allow us to improve the efficacy of this treatment. Our laboratory discovered that sCLU is induced by IR doses as low as 2 cGy
(2 rads), suggesting a role for sCLU in the cellular responses to ionizing radiation.
Understanding the processes involved in the induction of sCLU by IR will allow us to enhance the efficacy of radio-therapy and potentially screen for tumors that may be refractory to this type of treatment. Such a screen would allow an opportunity for molecular intervention to overcome these resistance mechanisms. The work described in this thesis characterized and elucidated the signal transduction pathways that regulate sCLU expression after IR exposure.
sCLU is thought to be a general stress responder, acting in a cytoprotective manner. One cytoprotective function of sCLU could be related to its ability to act as a molecular chaperone, preventing the precipitation of improperly folded proteins and aiding clearance of extracellular debris (Chapter 1). The experiments demonstrating sCLU’s ability to act in a manner similar to small heat shock proteins were performed in vitro, looking at the effects of the addition of exogenous CLU on various proteins after heat shock or oxidative stress (75, 406). It is intriguing to speculate on what role sCLU is performing as a secreted chaperone, since sCLU induction does not occur until several days after stress. It may be that sCLU is secreted from cells that survive cytotoxic stress,
125 and is needed to clear cellular debris from dying cells in order to “turn over” factors that could be potentially cytotoxic or inflammatory.
We found that CLU is transcriptionally repressed by the p53 tumor suppressor protein (Chapter 2). The p53 protein is stabilized in response to genotoxic stress and acts as a transcription factor for genes resulting in either cell cycle arrest or apoptosis. p53 can also act as a repressor of transcription, although the exact mechanism of repression varies. Several lines of evidence suggest that sCLU is transcriptionally repressed by p53:
(A) HCT116 colon cancer cells that are p53 null show a dramatic induction of sCLU after
IR as compared to cells that contain wild-type p53; (B) MCF-7 cells that contain the
HPV-16 E6 protein have high basal levels of sCLU as compared to cells without E6, and
(C) sCLU is induced after IR exposure in RKO cells that contain the HPV-16 E6 protein, but not in RKO parental cells that contain functional p53.
It has been suggested that sCLU is regulated through the cell cycle, with peak
induction seen as cells exit the G0 quiescent phase. Bettuzzi et al., demonstrated that
CLU message peaked in serum-starved normal human dermal fibroblasts as they entered
G0 (328). We examined sCLU expression during different phases of the cell cycle to account for the possibility that the transcriptional repression of sCLU by p53 was due to position in the cell cycle. Isogenic HCT116 cells that differ only in their p53 or p21 status were serum-starved and confluence arrested with the result that approximately 80%
of the cells arrested in G0. We noted little change in sCLU status in any of these cell lines as the cells progressed through the cell cycle, suggesting that repression of sCLU in cells with functional p53 protein was dependent on p53 and not on cell cycle phase.
126 The mechanism of CLU repression by p53 remains to be elucidated. There are several proposed models on how p53 can act as a transcriptional repressor (407). In the first model, p53 binds to its putative DNA binding sequence and sterically inhibits the binding of transcription factors required for induction. Upregulation of α-fetoprotein requires the binding of hepatic nuclear factor 3 (HNF-3) to its promoter region (333). A p53 binding site overlaps the HNF-3 binding site, thus when p53 is bound, HNF-3 is displaced. This model was also proposed to account for repression of Bcl-2 (332) and
HBV (334) genes by p53. In the second model, p53 binds and sequesters transcription factors required for transcriptional upregulation. For example, p53 can directly bind several transcription factors including Sp-1(335, 336), Ap-1 (337), NF-Y (338, 339),
Brn-3a (332) and C/EBPβ (340). In this regard, the similarities between Sp1 and Egr-1 transcription factors may be important and the possibility of p53 binding to, and repressing Egr-1 should be explored. p53 can also bind the TATA binding protein (TBP) in vitro and inhibit transcription by disrupting formation of the TFIID complex (341).
Additionally, p53 can repress the Map4 (408), α-tubulin (409) and survivin (410) genes through its interactions with the chromatin remodeling machinery. The ability of p53 to suppress survivin is of particular interest since as with sCLU, survivin is a potent pro- survival factor induced after stress. p53 associates with histone deacetylaces (HDACs) via an interaction with mSina and this interaction can be stimulated after DNA damage
(407). Alternatively, Johnson et al. have proposed a novel putative DNA binding sequence for p53 that is strictly involved in transcriptional repression (342).
Recently, our laboratory cloned a larger fragment of the CLU promoter (4250 bp) that contains a consensus p53 binding site. Using DNA pull down assays with a
127 biotinylated 4250 promoter, we have shown that p53 can bind to this site, and binding is disrupted 24-72 h after IR, which corresponds to the temporal induction of sCLU
(Leskov et al., unpublished data). This could provide a possible explanation for our data in Chapter 2, where p53 and sCLU protein levels are simultaneously high 48 h after IR in
MCF-7 cells. The p53 detected in these assays also shows a larger band that may correspond to monoubiquitinated p53. Grossman et al., demonstrate that MDM2 monoubiquitinates p53 and that p300 is required for further p53 polyubiquitination in vivo (411). It is interesting to speculate that MDM2 may be monoubiquitinating p53, which would destabilize p53 DNA binding without changing total p53 protein levels, resulting in the induction of sCLU. One could also speculate that p53 represses sCLU early after IR, due to its interaction with HDACs. After DNA repair is completed, p53 repression may be relieved via an MDM2 dependent mechanism, resulting in later elevated sCLU levels.
Our studies revealed that sCLU plays a role in cell survival after exposure to IR
(Chapter 3). To further understand cellular responses to IR, we investigated the signaling pathways required for the IR-induction of sCLU. We report that IGF-1R activation after
IR was required for sCLU induction. We demonstrated that IR-induced sCLU was dependent on a novel reactivation of the Src/Raf/Mek/Erk signaling cascade 24-72 h after
IR exposure, and that this signal culminated in the activation of the Egr-1 transcription factor (model, Fig. 3.7).
EGFR is over-expressed or constitutively activated in many types of tumors including colorectal, breast, pancreatic and ovarian cancers (397) and is known to be a mediator of radio-resistance in several tumor types including glioblastoma multiforme
128 and breast cancer cells through the activation of Erk-1/2 (398, 399). As a result, many therapies have been developed that specifically target EGFR including monoclonal antibody therapies and small molecule inhibitors that specifically target the kinase domain (355). Interestingly, the selective EGFR inhibitor, AG1478, did not block CLU promoter induction or regulate sCLU protein levels after 5 Gy (Fig. 3), nor did it affect
EGF-stimulated sCLU protein expression, possibly due to the involvement of other
EGFR family members.
IGF-1R is another membrane receptor shown to be upregulated after IR. IGF-1R activation results in mitogenic growth and cell survival (384), and Gooch et al., demonstrated that treatment of cells with IGF-1 could prevent doxorubicin and taxol induced apoptosis (400). Using AG1024, a selective inhibitor of IGF-1R, we were able to block sCLU induction after IR (Fig. 4), demonstrating the requirement for IGF-1R activation for IR-induced sCLU expression. In a recent report, it was shown that
AG1024 treatment of MCF-7 cells enhanced cell death after IR exposure (401). Our data strongly suggest that activation of IGF-1R may mediate cell survival effects through the downstream induction of sCLU.
MCF-7 cells produce and secrete IGF-1 under serum-free conditions (387). In addition, IGF-1R is often over-expressed in breast cancer (402). It was shown previously that peripheral lymph node stromal cells produce and secrete EGF and IGF-1, which can increase the growth of breast cancer cells (403). It is possible that EGF and IGF secretion by lymph nodes can induce the tumorigenesis of neighboring breast tissue, especially cells that have upregulated expression of EGFR or IGF-1R, through a paracrine mechanism. It is interesting to note that both EGF and IGF-1 were able to
129 induce sCLU expression (Fig. 3C). Additionally, serum starvation increased the basal activity of the CLU promoter compared to cells grown in whole serum, suggesting a possible autocrine feedback loop induced by IR, where irradiated cells not only up- regulate IGF-1R, but also presumably increase production of the ligand, IGF-1.
Consistent with a previous report (384), we demonstrate induction of IGF-1R after IR
(Fig. 4D), as well as a 20% increase in secretion of IGF-1, 24-72 h post-IR (Fig. 4E).
Importantly, the induction of IGF-1R and its ligand provide a plausible explanation for the late induction of CLU after IR, as well as the increase in basal promoter activity after serum-starvation.
We show, for the first time, that the Src-Raf-Mek-Erk-1/2 pathway is required for
IR-induced sCLU activation. This cascade culminates in the activation of the Egr-1 transcription factor. In addition, we demonstrate a novel re-activation of the MAPK cascade after IR that correlates with the temporal activation of sCLU. The physiological relevance of this biphasic activation of MAPK is unknown. EGFR may be required for the initial induction of MAPK after IR, and inhibitors to EGFR are known to potentiate the cytotoxicity of radiation therapy (404). In addition, Lu et al., demonstrated that increased IGF-1R production in MCF-7 cells caused increased resistance to Herceptin (a monoclonal antibody to the Her2/neu receptor) induced cell death (405), suggesting a role for IGF-1R signaling in the development of resistance to this type of therapy. Our data suggests that tumor cells that survive the initial phase of treatment may develop resistance to EGFR inhibitors, as a result of the late induction of MAPK signaling through IGF-R1 upregulation, and potentially the upregulation of sCLU. This suggests
130 that the current antibody and small molecule therapies used to treat EGFR positive tumors may be optimized by the addition of inhibitors to the IGF-1R pathway.
Recent data indicate that sCLU provides cytoprotection against doxorubicin, taxol and cisplatin in the treatment of cancer cells (63, 73, 74, 87). In this report, we show that sCLU provides cytoprotection in MCF-7 cells after IR exposure (Fig. 1). sCLU expression has been found to be elevated in many types of tumors, including prostate, colorectal and breast cancer (49, 51, 58). In a recent paper by Chen et al., it was shown that CLU message and protein were elevated in intestinal tumors derived from mice containing the Apc min (multiple intestinal neoplasia) mutation (59). IGF-1R and IGF-1 production are also elevated in many tumor types. Gleave et al., in a recent review, suggest using CLU and insulin-like growth factor binding proteins (IGFBPs) as targets for antisense therapy against prostate cancer (73), although they did not mention a possible connection between IGF-1R signaling and the induction of sCLU. Our data strongly suggests such a connection, which may explain the connection between elevated sCLU levels and the development of prostate cancer.
It is intriguing to speculate a possible role for sCLU in bystander effects, after radiation or chemotherapeutic therapies. An increased production and secretion of sCLU by tumor cells into the lymph or vasculature system may provide a survival effect for neighboring or metastatic cancer cells. Additionally, secretion of IGF-1 by normal or tumor cells may provide a means for sCLU upregulation.
The signaling pathway that relieves p53 repression, allowing for sCLU induction after IR, has not yet been determined. Recently, in a paper by Lu et al., it was shown that
Src family kinases could inhibit the function of PTEN (412). PTEN has been shown to
131 be an inhibitor of IGF-1R activated MAPK (413) as well as the Akt signaling pathway
(412). One function of Akt is to stabilize Mdm2 allowing for degradation of p53.
Additionally, Tanno et al., have shown that Akt activation can up-regulate IGF-1R (414).
The cross-talk between the IGF-1R, PTEN and Akt pathways may provide an intriguing connection between the signaling cascade resulting in sCLU induction after IR and the repressive effects of p53 (Figure 4.1). Preliminary data from our laboratory support a role for PTEN and Akt in the regulation of sCLU. Over-expression of a constitutively active Akt or expression of a catalytically dead PTEN in 1403 cells resulted in significantly higher basal levels of CLU promoter activity as compared to cells transfected with vector alone (Criswell et al., unpublished data).
The exact regions of the CLU promoter that are required for IR induction have not yet been determined. We demonstrated that Egr-1 was required for sCLU induction, but there are three potential Egr-1 binding sites within the 1403 promoter fragment. Deletion analyses and point mutations of the CLU promoter will allow us to identify the sites required for IR-induction, as well as sites required for p53 repression. These experiments will also be useful in determining if the induction of sCLU by other cytotoxic agents occurs through the same signaling pathways as those induced by IR. Determining the exact element required for IR induction will allow us to potentially utilize the CLU promoter for combination gene targeting and radiation therapies. The Egr-1 promoter linked to the herpes simplex virus thymidine kinase gene is currently being utilized in this manner to sensitize tumor cells for radio-therapy (285, 415, 416). The CLU promoter IR- inducible element may be more useful since it is induced by much lower doses of IR and because it is regulated by p53. Since p53 is mutated in over 50% of all human tumors,
132 using the IR-inducible 4250 CLU promoter with the p53 binding site will allow us to target this construct to be active in the tumor cells missing p53, while sparing the normal cells that still contain wild-type p53.
This thesis has described the regulation of sCLU after IR. These studies shed light on the cellular responses to ionizing radiation. One of the problems facing cancer patients undergoing radio-therapy is the development of refractory cells/tumors to this treatment. sCLU may play a role in the development of radio-resistance, since cells lacking sCLU tend to be more sensitive to IR. Targeting the pathways that result in sCLU induction should sensitize tumor cells to radio-therapy. A greater understanding of these processes will allow for the improvement of cancer treatment.
133 IGF-1R
Src PI-3K IRS-1 SOS PTEN Grb2 Ras Raf Akt
MEK-1
Erk-2 Mdm2 Erk-1
Erk-2 Erk-1 p53 Egr-1 CLU promoter
Figure 4.1. Model depicting potential cross-talk between the IGF-1R dependent signaling pathway leading to sCLU induction after IR and a potential pathway signaling to the p53 transcriptional repression of sCLU.
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